Elution Research Articles

A-070 Evaluation of Plain and Serum-Separator Blood Collection Tubes for the Measurement of Steroid Hormones by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)

Abstract Background Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) is the preferred method for quantifying steroids. Commonly measured steroids 11-deoxycortisol, androstenedione, testosterone, 17-hydroxyprogesterone and dehydroepiandrosterone (DHEA). Although serum separator blood collection tubes may not be recommended but their frequent use is attributed to the ease of serum separation. Moreover, the serum separator tube may be the only available option for analysis, without the need for additional sample collection. Under such conditions, from certain serum separator tubes, we observed occasional interference in LC-MS/MS steroid method. We pursued to evaluate the performance of blood collection tubes without and those with serum separators, for the quantification of specified steroids by LC-MS/MS. Methods Blood samples were collected from 10 volunteers using both plain and serum separator blood collection tubes procured from two major suppliers (Greiner and BD). The specified steroids were measured by LC-MS/MS method. The procedure involved addition of labeled internal standards to the samples and extraction of steroids in methyl tert-butyl ether. Subsequently, the extract dried under a stream of nitrogen, and the residue was reconstituted in the mobile phase. LC-MS/MS analysis involved multiple reaction monitoring (MRM) for the specified steroids and their internal standards. Results The figure shows a sample chromatogram from a subject with blood samples collected in a plain (A) and serum separator tubes from Greiner (B) and BD (C1 and C2, a zoomed of C1). In this sample, from the plain tube, the concentrations of 11-deoxycortisol, androstenedione, testosterone, 17-hydroxyprogesterone and DHEA were 27, 55, 690, 99 and 283 ng/mL respectively. No significant interference was observed in Greiner serum separator tube. Whereas BD serum separator tube exhibited several erroneous peaks making the quantitation of several steroids challenging or inaccurate. Conclusions In the described LC-MS/MS method, BD serum-separator tubes exhibited erroneous peaks. These peaks were not seen in Greiner serum-separator tubes.

B-248 Enhanced Detection of of Hemoglobin Variants in Clinical Research using Dried Blood Spot and HRAM MS

Abstract Background Hemoglobinopathies are one of the most prevalent genetic disorders, with more than 330,000 affected births occurring annually. Common techniques used in Hb analysis are electrophoretic and chromatographic assays. However, there are challenges to differentiate isomers (i.e., between Hb C and Hb E), very similar masses, or new variants due to their lack of resolution. Mass spectrometry (MS) gained popularity in clinical research because of its robustness and versatility in analyzing a wide range of samples and analytes from small molecules to proteins. Here, we present the top-down analysis for enhanced detection of various hemoglobin variants using Orbitrap Exploris for clinical research. Methods Proteins were directly extracted from dried blood spots (DBS) and analyzed by Thermo Scientific™ Orbitrap Exploris™ 240 with BioPharma Option coupled to Vanquish HPLC system. A 3.2 mm disc was punched from the DBS cards to 96 well plate and 50 uL water was added to re-hydrate. For protein precipitation, 150 uL acetonitrile was added and incubated at -20 ⁰C for 15 minutes. After micro-centrifugation, 160 uL of supernatant was removed and 70 uL water was added to resuspend. For LC-MS analysis, 10 uL of supernatant was diluted 8 times with mobile phase A and transferred to another 96-well plate. LC separation was performed on MAbPac RP column 4 um, 1 x 100 mm, and data-dependent acquisition mode was operated. Results With direct protein extraction from DBS, the entire sample preparation time is less than 1 hour. The MAbPac column showed its capability of separating between different chains with minor overlap between normal beta and HbS beta chains. Data processing was performed by ProSightPD, resulting in ∼45% sequence coverage for both normal Hb and HbS beta chains. The mass difference between normal beta and Hb S beta chains is about 30 Da, which results in about 1.6 m/z difference at a charge state of 19. This mass difference can often be hindered by other proteins or interferences from the matrix if the mass analyzer doesn’t have sufficient resolving power. Here we successfully applied Orbitrap resolution at 120,000 to isolate these similar masses for accurate variants or subunits identification including normal alpha, normal beta, Hb S, and delta chains. The confirmation of the delta chain can be beneficial since its abundance is usually less than 0.5% in adults, which shows potential for detecting low abundant variants using this method. Also, the calibration curve was generated to demonstrate the method's feasibility which is not a typical quantitation method for hemoglobinopathies. For quantitation, three most abundant m/z values from 2 charge states were added to TraceFinder for peak area extraction and normal Hb beta chain was used as an alternative internal standard for data processing. The 3-day inter/intraday analysis generated great reproducibility with CV < 10% and LOQ was determined to be 2.5 mg/mL with R2 values >0.994. Conclusions Successful demonstration of a single-step protein extraction directly from DBS and top-down analysis of Hb variant for clinical research.

B-025 Revolutionizing NGS-driven Laboratory Developed Tests: The impact of automated liquid handling on plate-based bead clean-up

Abstract Background Automated liquid handling systems are proving to be a transformative technology in the landscape of Laboratory Developed Tests (LDTs), especially those utilizing Next-Generation Sequencing (NGS). By enhancing accuracy, boosting efficiency, and offering scalable solutions, these systems enable labs to meet current and future challenges more effectively. The right system not only improves the quality of diagnostics but also positions labs to be agile and adaptive in a rapidly advancing healthcare environment. firefly® is a well-suited liquid handling solution for diagnostics labs developing LDTs due to its compact footprint, user-friendly software and exceptional versatility. Validated protocol templates that support NGS library preparation are readily available from the firefly cloud and can be adapted to meet specific lab needs. For example, automated bead-based clean-ups designed for 96- and 384- NGS applications are available on firefly. These remove DNA fragments of undesirable sizes, enzymes or primers rom the amplified DNA after a PCR reaction. Automating this process facilitates higher throughput and scaling. firefly has a unique combination of 6 positive displacement dispense heads and a 96/384-well air-displacement pipetting head, making it ideal to distribute the reagents required for bead clean-ups. Methods firefly protocols were developed and optimized to be compatible with a range of elution volumes and bead ratios. Protocol performance was tested using library DNA (∼300bp). The same input DNA was plated out and quantified using the QuantiFluor dsDNA system (Promega) before and after running the protocol. This data was used to assess any reduction in DNA concentration and uniformity across the plate. The protocol's ability to successfully modulate the fragment size distribution was demonstrated using a 50bp DNA ladder and different bead ratios. Data was obtained using a Fragment Analyzer (Agilent, DNF-474 HS NGS Fragment Kit). Well-to-well contamination was assessed by running no-template-controls (NTCs) in wells adjacent to library DNA, then analyzed by qPCR using a LightCycler 480 System (Roche, KAPA Library Quantification kit). Results 96 format has a setup time of 10 minutes, run time of 43 and %CV library concentration of <6%. 384 format has a setup time of 10 minutes, run time of 47 and %CV library concentration of <8%. Uniformity of concentration and fragment sizes was seen across the plates. Comparison of the library concentration before and after the clean-up showed a 5% reduction in DNA concentration after the 96-well plate clean-up and an 11% reduction in library concentration when using the 384-well plate clean-up. Fragment size analysis showed that fragment sizes can be successfully modulated on firefly by changing the bead ratio of the firefly run and no detectable well-to-well contamination was detected by qPCR. Conclusions We have demonstrated that automated bead clean-ups on firefly are uniform show uniformity across both 96- and 384-well plates, with no detectable well-to-well contamination, to deliver the consistent and robust results required for an LDT. Bead-based cleanups on firefly offer a fast, 100% walkaway solution. By running 96-samples at once on firefly, we see approximately a 6-fold increase in throughput than when performed manually by a single user.

Using artificial neural networks to elucidate retention interactions on stationary phases with amine moieties dedicated to supercritical fluid chromatography

The retention mechanisms of small molecules on a wide range of stationary phases using different mobile phase compositions are one of the most discussed topics in current state-of-the-art supercritical fluid chromatography (SFC). Although various theories have been published on retention behavior, prediction, and stability over time, an in-depth insight into the mechanisms is still lacking. Our study focused on the description of the retention mechanisms on columns with amine moieties, i.e., 2-ethylpyridine, 2-picolylamine, 1-aminoanthracene, and diethylamine, dedicated to SFC, using three different compositions of organic modifier, i.e., methanol, methanol + 10 mmol/L NH3, and methanol + 2 % H2O, in CO2. An extensive set of analytes characterized by more than 200 molecular descriptors was used. The importance of the effect of each molecular descriptor on the retention behavior was determined by artificial neural networks. Thus, the descriptors increasing and/or decreasing retention on individual columns using different mobile phase compositions were defined. The data collected over one year of column use were statistically evaluated to describe the retention behavior of small molecules on the tested stationary phases using three organic modifiers and to describe the changes in retention behavior over time.As the development of the SFC method can be laborious and time-consuming, this publication offers a detailed description of the interactions taking place between the analyte and the SFC stationary phase. Understanding these fundamental processes will enable faster development of SFC methods using quality-by-design principles.

B-176 Rapid Diagnosis of 21-Hydroxylase Deficiency Using LC-MS/MS for Urinary Unconjugated Steroid Analysis

Abstract Background Urinary steroid profiling through gas chromatography-mass spectrometry (GC-MS) serves as a potent diagnostic tool for detecting abnormal steroid production linked to congenital enzyme deficiencies and steroid-related diseases. This technique involves deconjugating and extracting both conjugated and unconjugated steroids, followed by derivatization and analysis via GC-MS. It provides comprehensive insights into steroid metabolism, aiding in the identification of genetic disorders and conditions with atypical steroid levels. However, the procedure is intricate and time-consuming, particularly the deconjugation of steroids for analysis. The present study aimed to assess the usability of LC-MS/MS assays measuring unconjugated Δ4- and Δ5-steroids in urine for diagnosing steroid abnormalities. Methods The Biotage ISOLUTE SLE+ (Biotage, Sweden) was used for sample preparation, with dichloromethane for elution. LC-MS/MS analysis was performed on an LCMS8060NX (Shimadzu, Kyoto, Japan), using a Shim-Pack Velox Biphenyl column 2.1mm*150mm* 1.8μm (Shimadzu GLC, Tokyo, Japan). The mobile phase consisted of a 0.2mmol/L ammonium fluoride aqueous solution and a 0.2mmol/L ammonium fluoride methanol solution. We targeted 38 steroids, including four steroid medications, and used 19 stable isotope-labeled steroids for quantification. For the measurements, 30 neonatal urine samples were used (IRB Approval Number 20221210), which were suspected of 21-hydroxylase deficiency (21OHD) based on clinical symptoms or screening for 17α-hydroxyprogesterone (17OHP) using Dried Blood Spot. Of these, 18 had been confirmed as 21OHD cases by exceeding the GC-MS diagnostic criterion for Pregnanetriolone (>0.06ng/g Creatinine). Results Blood 21-deoxycortisol (21DOF) is a reliable marker for diagnosing 21OHD, which is generated from 17OHP through an alternative biochemical route. In consideration of that, we measured the urinary concentration of unconjugated 21-deoxycortisone (21DOE, precurcer of Pregnanetriolone), into which 21DOF is putatively converted in the kidney. In cases where 21OHD was excluded by GC-MS diagnosis, 21DOE was undetectable, falling below the sensitivity levels. On the other hand, 21DOE was detected in all samples confirmed 21OHD cases, consistent with the conversion of 21DOF to 21DOE in the kidney. Conclusions The present study suggested the usability of LC-MS/MS assays measuring urinary unconjugated 21DOE for diagnosing 21OHD. This approach provides a faster, less complex method for diagnosis, eliminating the need for the deconjugation process. To establish the value of urinary unconjugated steroids as diagnostic markers for steroid abnormalities, efforts to accumulate further findings are underway using clinical samples.

B-182 Development and validation of an LC-MS/MS assay for simultaneous measurement of serum androstenedione and 17-hydroxyprogesterone in serum

Abstract Background Androstenedione and 17-hydroxyprogesterone are measured via immunoassays or high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) in clinical laboratories. Immunoassays have limited sensitivity and specificity for certain steroid hormones testing. We aimed to develop and validate a sensitive, accurate, and specific in-house LC-MS/MS assay to simultaneously measure both 17-hydroxyprogesterone and androstenedione in serum. Methods 13C-labeled androstenedione and 17-hydroxyprogesterone internal standards were added to calibrator, control, and patient samples. A hexane/ethyl acetate solution was used in a liquid-liquid extraction procedure. The organic layer was then removed and dried followed by reconstituting the extracts in 2.5 mM ammonium formate in 50% methanol. The HPLC mobile phase gradient began with 47%, 2.5 mM ammonium formate in water and 53%, 2.5 mM ammonium formate in methanol. This progressed to 56% of the methanol phase over three minutes. The mixtures were separated though an Agilent 1260 Infinity HPLC system with a Poroshell 120 EC-C18 analytical column (2.1 × 50 mm × 2.7 µm). The separated HPLC eluents then entered an Agilent 6460C QQQ mass spectrometer in positive ion mode through an electrospray ionization source where protonated androstenedione (m/z = 287.2) with the protonated androstenedione-[2, 3, 4-13C3] internal standard (m/z = 290.2) and protonated 17-hydroxyprogesterone (m/z = 331.2) with the protonated 17-hydroxyprogesterone-[2, 3, 4-13C3] (m/z = 334.2) were mass-selected for collision-induced dissociation. For androstenedione, respective quantifier and qualifier m/z values of 287.2 > 97.1 and 287.2 > 109.1 were used while respective quantifier and qualifier m/z values of 331.2 > 97.1 and 331.2 > 109.1 were used for 17-hydroxyprogesterone. Validation studies were performed including imprecision, lower limit of quantitation (LLOQ), analytical measurement range (AMR), interference, specificity, carryover, extraction recovery, matrix effect, and comparison with a reference laboratory LC-MS/MS method. The total allowable error limits used in this study were 23.5% for androstenedione and 30.2% for 17-hydroxyprogesterone. Results Total imprecision results across three concentrations yielded a maximum CV of 4.6 % for androstenedione and 6.5% for 17-hydroxyprogesteron. The AMRs of androstenedione and 17-hydroxyprogesterone were 0.2 to 13.4 and 0.1 to 22.0 ng/mL without significant carryovers. No significant interferences were observed from 11-deoxycorticosterone (700 ng/dL), testosterone (2500 ng/dL), DHEA (1000 ng/mL), bilirubin (30.1 mg/dL), hemoglobin (1032 mg/dL), or triglycerides (1858 mg/dL). The extraction recoveries of androstenedione and 17-hydroxyprogesterone were 98% and 81%, respectively. No significant matrix effects were identified. In comparison to the reference lab LC-MS/MS method, the in-house LC-MS/MS assay demonstrated a 0.523% bias for androstenedione and a -3.77% bias for 17-hydroxyprogesterone with both exhibiting excellent correlations with the reference lab measurements. More specifically, our results (as a function of values obtained from the reference lab) followed the relationship of y = 1.039x - 0.092 (R = 0.9951) for androstenedione and y = 0.957x + 0.01 (R = 0.9974) for 17-hydroxyprogesterone. Conclusions An LC-MS/MS assay is developed for simultaneous detection of serum androstenedione and 17-hydroxyprogesterone. The assay demonstrates acceptable imprecision, AMR, and accuracy. No significant carryover and interference from other structurally similar steroid hormones and other common interferents are identified.

B-168 Development and Validation of a Quantitative Ultra-Performance Liquid Chromatography Quadrupole Time-of-Flight (UPLC-QToF) Method for Urine Organic Acid Analysis

Abstract Background Urine organic acid (UOA) analysis is essential for the diagnosis of inborn errors of metabolism (IEMs). Traditionally, UOA analysis is performed with gas chromatography-mass spectrometry (GC-MS) and requires time-consuming sample preparation steps including liquid-liquid extraction and derivatization. The rapid development of Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS) in the past few years provides the opportunity to perform UOA analysis with a dilute-and-shoot methodology. We describe the development and validation of a quantitative Ultra-Performance Liquid Chromatography Quadrupole Time-of-Flight (UPLC-QToF) method for UOA analysis. Methods Urine specimens were diluted to normalize creatinine concentrations to 1 mmol/L. 20 µL of urine specimen (diluted), calibrator, or quality control (QC) material was mixed with 400 µL of mobile phase A (0.05% formic acid in water) and a mixture of isotope-labeled internal standards. After centrifugation, 10 µL of the supernatant was analyzed using a Xevo G3 QTOF mass spectrometer (Waters) with a ACQUITYTM Premier HSS T3 1.8 µm VanGuardTM FIT 2.1 x 150 mm column (Waters). Data collection was performed with negative electrospray ionization (ESI) mode using the MSE method to produce fragment ions when applicable. Repeatability, reproducibility, and carryover were assessed using the QC materials. The analytical measuring range (AMR) was assessed using synthetic urine spiked with increasing concentrations of each organic acid. Accuracy was assessed by method comparison with the UOA test performed at Mayo Clinic Laboratory and by spike-recovery study using a pooled urine specimen. Matrix effect was also evaluated with matrix dilution study. Results An optimized LC method was used to enable high-resolution separation of selected UOAs (N = 29) and isomers. Total analytical time was 20 min per injection. Both linear and quadratic regressions were used to build the calibration curves. AMR and correlation coefficients of a few representative UOAs were: orotic acid (3.4 to 214.2 mmol/mol creatinine, R^2 = 0.99, linear regression); 2-methylcitric acid (4 to 189 mmol/mol creatinine, R^2 = 0.99, linear regression); 3-methylcrotonylglycine (0.3 to 18.0 mmol/mol creatinine, R^2 = 0.99, linear regression). Repeatability and reproducibility were mostly <=10% CV and no carryover was observed. Spike-recovery study demonstrated recoveries between 80% and 120%, and method comparison study demonstrated no discrepancies with results from Mayo Clinic Laboratory. Conclusions We have developed and validated a novel UPLC-QTOF method for UOA analysis to support the diagnosis of IEMs with acceptable analytical and clinical performances. Compared with the traditional GC-MS method, the UPLC-QTOF method requires a very small specimen volume and does not require laborious and time-consuming sample preparation steps. Continued optimization of the method will be pursued to measure more UOAs to support the diagnosis of more IEMs.

Determination and antioxidant analysis of seven flavonoids in bamboo-leaf extracts

The flavonoid contents of different bamboo-leaf extracts and their relationships to antioxidant activity were investigated in this study by preparing nine samples using two commercially available bamboo-leaf extract products and seven bamboo-leaf extracts such as Phyllostachys edulis. A high performance liquid chromatography (HPLC) method was established to determine seven flavonoid components (orientin, isoorientin, vitexin, isovitexin, tricin, luteolin and luteoloside) in these samples, which were separated using a SymmetryShieldTM RP8 column (250 mm×4.6 mm, 5 μm) under gradient-elution conditions using acetonitrile as mobile phase A and 0.5% (v/v) acetic acid aqueous solution as mobile phase B. The antioxidant activities of the samples were evaluated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radical-scavenging assays, with half inhibitory concentration (IC50) as an indicator and the butylated hydroxytoluene (BHT) and tert-butylhydroquinone (TBHQ) antioxidants as positive controls. Pearson correlation was then used to analyze the relationship between flavonoid content and antioxidant activity. The HPLC method was found to be accurate and reliable for determining the flavonoid contents of the bamboo-leaf extracts. The seven flavonoids were well separated, and good linear relationships were exhibited (correlation coefficients (R2)≥0.9990). Furthermore, the contents of the seven flavonoids in the bamboo-leaf extracts ranged from 14.97 to 183.94 mg/g, with the highest content of 183.94 mg/g recorded for Phyllostachys edulis. The bamboo species exhibited significantly different flavonoid contents, with Phyllostachys edulis showing the highest orientin, isoorientin, and vitexin levels of 38.45, 101.30, and 9.42 mg/g, respectively. Moreover, the bamboo-leaf extracts exhibited IC50 values of 78.23-179.41 mg/L for DPPH-radical-scavenging, while values of 203.48-1250.81 mg/L were recorded for hydroxyl radicals. The Phyllostachys edulis leaf extract exhibited the strongest antioxidant activity, with the lowest IC50 values of 78.23 and 203.48 mg/L for DPPH and hydroxyl, respectively; it showed greatly significant for the further development and application of Phyllostachys edulis. Finally, the relationships between flavonoid content and the DPPH- and hydroxyl-radical-scavenging activities (based on the IC50 values) were correlated, which revealed that the orientin and isoorientin contents are closely related to the antioxidant activities of the bamboo-leaf extracts. Consequently, the orientin and isoorientin contents can be used as indicators for evaluating the antioxidant activities of bamboo-leaf extracts.

Accurate determination of three new antihypertensive drugs illegally added to food using ultra-high performance liquid chromatography-tandem mass spectrometry

Effective strategies are required to address food safety issues related to the illegal addition of antihypertensive drugs to food and claims of antihypertensive function. In this study, a novel ultra-high performance liquid chromatography-triple-quadrupole mass spectrometry (UHPLC-MS/MS) method was developed for the simultaneous determination of three antihypertensive drugs (azilsartan, candesartan cilexetil, and lacidipine) in 12 food matrices (pressed candies, solid beverages, alternative teas, tea drinks, biscuits, jellies, mixed liquors, oral liquids, medicinal teas, tablets, hard capsules, and soft capsules). Initially, mass spectrometry parameters, such as the collision energies of the three antihypertensive drugs, were optimized. Subsequently, the response intensities and chromatographic separation conditions of the three drugs in different mobile phases were compared. In addition, to enhance the recoveries, various extraction solvents and purification methods, including solid-phase extraction (SPE) columns and the QuEChERS technique, were investigated. In the developed method, sample determination involved three steps. First, the sample was extracted using 0.2% (v/v) formic acid in acetonitrile and then filtered using high-speed centrifugation, in addition, the extracted solution of alternative tea and medicinal tea was purified using the QuEChERS technique. Second, the supernatant was diluted with water, and filtered through a 0.22 μm polytetrafluoroethylene (PTFE) membrane. Finally, the analytes were separated on an Agilent Eclipse Plus RRHD C18 column (50 mm×2.1 mm, 1.8 μm) using a 5 mmol/L ammonium formate aqueous solution and acetonitrile as the mobile phases under gradient elution conditions and then detected using UHPLC-MS/MS with positive electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode. Quantitative analysis was performed using a matrix-matched external standard method. Methodological validation showed good linear relationships for all three antihypertensive drugs in the investigated concentration ranges, with correlation coefficients (r2) greater than 0.996. The limit of detection (LOD) and limit of quantification (LOQ) of lacidipine were 0.02 mg/kg and 0.04 mg/kg, respectively, whereas those of the other two drugs were 0.01 mg/kg and 0.02 mg/kg, respectively. In the 12 food matrices, the average recoveries of the drugs ranged from 86.6% to 107.5% with relative standard deviations (RSDs) of 1.1%-10.9% (n=6) at low, medium, and high spiked levels. Furthermore, this method was successfully applied to the analysis of real food samples. Overall, the newly developed method is simple, rapid, sensitive, accurate, and suitable for the qualitative and quantitative determination of antihypertensive drugs in different food matrices. This work could provide technical support for food safety agencies in implementing measures against the illegal addition of antihypertensive drugs to food.

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

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

THE INDUSTRIAL IMPORTANCE OF TECHNOLOGY TRANSFER FOR ANALYTICAL METHOD DEVELOPMENT AND VALIDATION–APPLICATION TO VILAZODONE HYDROCHLORIDE DOSAGE FORM

Objective: The research aims to create a fast, economical, and efficient tech transfer method for analyzing bulk materials and pharmaceutical dosage forms. Methods: The analysis was performed using an Acquity UPLC BEH C18 analytical column (130 Å, 1.7 µm, 3 mm x 100 mm) with detection of the analyte at 238 nm. The mobile phase flow rate was optimized at 0.3 ml/min, and an injection volume of 0.5 µl** was used. The isocratic mobile phase consisted of a blend of buffer (0.1% OPA) and acetonitrile at a ratio of 40:60% v/v. Results: All validation parameters of the analytical method were acceptable. The analyte eluted at 3.08 min with satisfactory theoretical plates and a tailing factor. The recovery of the analyte was 99-100%, and the method's precision was 0.59 and 0.72% RSD. The technique showed no interference, indicating specificity. The linearity was demonstrated over the 20-60 µg/ml range, with a regression coefficient of 0.999. Conclusion: The developed method is cost-effective, specific with minimal interference, and time-saving. It also reduces solvent consumption for quality control analysis of vilazodone hydrochloride samples. The method demonstrates good linearity, robustness, and accuracy.

Validated high performance thin layer chromatographic method for simultaneous quantification of betulinic acid, β-sitosterol and lupeol in fruits, leaves, root bark and stem bark of Dillenia indica Linn

AbstractDillenia indica is one of the important medicinal plants and is extremely popular in many systems of medicine including ayurvedic, homeopathic, and siddha. Traditionally, different parts of this plant have been used to treat cancer, wound healing, diabetes, diarrhea, bone fractures, abdominal pains, cuts and burns. In the present study, a simple, precise, accurate and validated high performance thin layer chromatographic (HPTLC) method was developed for simultaneous estimation of three pharmaceutically active compounds i.e., betulinic acid (BE), β-sitosterol (BT) and lupeol (LP) in fruits, leaves, root bark and stem bark of D. indica. Standards and plant samples were applied on TLC aluminum plate precoated with 0.2 mm layer of silica gel 60F254. The plate was run in a twin glass chamber comprising toluene:methanol:chloroform (8:1:1, v/v/v) as a mobile phase which resulted in better separation of compounds. The plates were immersed in anisaldehyde-sulfuric reagent and then heated at 105 °C for 5 min in CAMAG TLC plate heater for appearance of bands. Densitometric scanning was performed at λmax = 525 nm using tungsten light source in CAMAG TLC Scanner4 armed with WinCATS software. RF values of BE, BT and LP standards and those of plant samples were found to be 0.38 ± 0.01, 0.54 ± 0.01 and 0.65 ± 0.02 respectively. The method was further validated by following the International Conference of Harmonization (ICH) guidelines. For BE, BT and LP, the linear regression data for the calibration plots revealed a satisfactory linear association with correlation coefficients (r2) = 0.9736, 0.9989 and 0.9957, respectively. Linear ranges for BE, BT and LP were 2,000–6,000 ng/band, 200–1,000 ng/band, and 200–600 ng/band respectively. Accuracy of the method was assessed by recovery study conducted at three different levels with the average recovery of 99.19, 99.69 and 100.95% for BE, BT and LP, respectively. The results exhibited the highest content of BE (0.148 ± 0.01%), BT (0.031 ± 0.01%) and LP (0.047 ± 0.01%) in stem bark. The developed method will be useful for routine and quality control analysis of fruits, leaves, root bark and stem bark of D. indica species.

Whiteness and greenness assessments of a sensitive HPLC method with fluorimetric detection for dapagliflozin quantitation in human plasma: Application to a healthy human volunteer.

The evident ecological impact of human actions, like air pollution, global warming, and ozone depletion, underscores the need for environmentally friendly approaches across various domains, including analytical chemistry. This study aimed to establish a validated, eco-friendly, and sustainable approach utilizing a fluorescence detector coupled with high-performance liquid chromatography for quantifying the antihyperglycemic agent dapagliflozin (DAPA), in human plasma. This method employed a C18 Microsorb MV (4.5 × 250 mm, 5 μm [particle size]) column at 40°C, with 40:60% v/v isocratic elution of acetonitrile and (0.1%) orthophosphoric acid as the mobile phase at 1 mL/min flow rate. DAPA and the internal standard demonstrated their greatest response by performing excitation at 225 nm (λex) and recording chromatograms at an emission wavelength (λem) equal to 305 nm. The presented approach demonstrated high linearity between 50 and 2000 ng/mL and full adherence to the guidelines of the US Food and Drug Administration regarding the validation of bioanalytical methods. The described technique was effectively used for quantification of DAPA in human plasma samples from a healthy male participant who received a tablet of 10 mg DAPA. Analytical Eco-Scale, Analytical GREEnness metric, and the recently created ChlorTox Scale were utilized for greenness assessment. Additionally, the "Red, Green, and Blue 12" model was used in whiteness evaluation.

Development of an untargeted DNA adductomics method by ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry

Genotoxicants originating from inflammation, diet, and environment can covalently modify DNA, possibly initiating the process of carcinogenesis. DNA adducts have been known for long, but the old methods allowed to target only a few known DNA adducts at a time, not providing a global picture of the “DNA adductome”. DNA adductomics is a new research field, aiming to screen for unknown DNA adducts by high resolution mass spectrometry (HRMS). However, DNA adductomics presents several analytical challenges such as the need for high sensitivity and for the development of effective screening approaches to identify novel DNA adducts. In this work, a sensitive untargeted DNA adductomics method was developed by using ultra-high performance liquid chromatography (UHPLC) coupled via an ESI source to a quadrupole-time of flight mass spectrometric instrumentation. Mobile phases with ammonium bicarbonate gave the best signal enhancement. The MS capillary voltage, cone voltage, and detector voltage had most effect on the response of the DNA adducts. A low adsorption vial was selected for reducing analyte loss. Hybrid surface-coated analytical columns were tested for reducing adsorption of the DNA adducts. The optimized method was applied to analyse DNA adducts in calf thymus, cat colon, and human colon DNA by performing a MSE acquisition (all-ion fragmentation acquisition) and screening for the loss of deoxyribose and the nucleobase fragment ions. Fifty-four DNA adducts were tentatively identified, hereof 38 never reported before. This is the first untargeted DNA adductomics study on human colon tissue, and one of the few untargeted DNA adductomics studies in the literature reporting the identification of such a high number of unknowns. This demonstrates promising results for the application of this sensitive method in future human studies for investigating novel potential cancer-causing factors.

Advanced stability-indicating RP-HPLC method for the quantification of lurasidone hydrochloride in bulk and PLGA-based in situ implant formulation

The aims of the present investigation was to develop and validate stability-indicating RP-HPLC method for the estimation of Lurasidone hydrochloride (LURA-H) followed by its drug product, LURA-H encapsulated poly-D,L lactic-glycolic acid (PLGA) based in-situ depot forming implant (LURA-H-PLGA-ISI). The LURA-H-PLGA-ISI formulation was developed by simple mixing method. According to international conference on harmonization guidelines, RP-HPLC method was developed and validated using Waters 2695 and discovery C18, 5μ, 250×4.6 mm ID column. Force degradation studied were performed by various degradation techniques. The chromatographic separations of LURA-H as well as LURA-H-PLGA-ISI with good resolutions have been achieved using the mobile phase 0.1% orthophosphoric acid and acetonitrile (50:50). The linearity in the range of 25-150 μg/mL of developed method. LOD and LOQ limits for LURA-H were found to be 0.07µg/mL and 0.22 µg/mL, respectively. The % RSD was found to be <2% showing the precision of developed method. The accuracy of developed method was demonstrated which is close to 100±2%. Little modifications in the chromatographic conditions indicated robustness of the developed method. Further, solution stability of LURA-H and LURA-H-PLGA-ISI was stable at room temperature. Furthermore, force degradation studies demonstrated LURA-H was unaffected and stable under thermal, photodegradation and neutral (hydrolytic) stress conditions. AGREE and GAPI assessment demonstrated the developed method is environmentally sustainable. The developed method is simple, robust, precise, accurate and sensitive which can be utilized for the regular analysis of LURA-H in quality control laboratories of bulk drug substance and PLGA containing formulations of LURA-H.

Comparison of piperine content, antimicrobial and antioxidant activity of Piper chaba root and stem

Piper chaba (locally known as “Choi Jhal”) is used traditionally as spices and folk medicine in different parts of Bangladesh. One of the most important bioactive compounds in this plant is piperine. In this study, the amount of piperine in P. chaba root and stem was investigated and the optimal solvent for piperine extraction at room temperature was also studied. High performance liquid chromatography (HPLC) was operated using a reverse phase column where methanol and water (70:30) were used as mobile phase. The detection was performed using photo diode array (PID) detector at a wavelength of 345 nm. The standard piperine showed linearity between 0.005 % and 0.04 % and the correlation co-efficient found for the linearity was 0.9933. The percentage of relative standard deviation (RSD) for both retention time and peak area were less than 2.0 %. The theoretical plate number (N) > 3000 and a tailing factor (T) < 1.5 were found in the acceptable range. The recovery percentage (%) of standard piperine was 99.16 %. Low value of co-efficient of variation and standard deviation are recognized for high precision of the method. The highest amount of piperine was found in root extracted with methanol (MR) amounting to 1.75 % in the root powder. The maximum amount of piperine in the stem was 1.59 % when extracted with methanol (MS). The piperine quantification in other extract like n-hexane root (HR), ethyl acetate root (ER), n-hexane stem (HS), ethyl acetate stem (ES) were 0.76 %, 1.69 %, 0.33 % and 1.46 % respectively. Methanol has given the highest yield of piperine compared to ethyl acetate and n-hexane for both root and stem. The developed method was simple, rapid, economic and validated in terms precision, accuracy and recovery. This selective method is found to be repeatable, accurate and successfully utilized for the Piper extract in marketed and pharmaceutical samples with well chromatographic conditions. The ethyl acetate extract of root and stem showed promising DPPH (1, 1-diphenyl-2-picrylhydrazyl) free radical scavenging activity with an IC50 value of 39.62 ± 0.95 μg/mL and 43.85 ± 1.50 μg/mL respectively. The study reports potential antibacterial activity and antifungal activity of P. chaba root and stem extracts. These outcomes revealed that different extracts of P. chaba may be used as natural preservatives.

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

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

Multiscale simulation of liquid chromatography: Effective diffusion in macro–mesoporous beds and the B-term of the plate height equation

We performed multiscale simulations of analyte sorption and diffusion in hierarchical porosity models of monolithic silica columns for reversed-phase liquid chromatography to investigate how the mean mesopore size of the chromatographic bed and the analyte-specific interaction with the chromatographic interface influence the analyte diffusivity at various length scales. The reproduced experimental conditions comprised the retention of six analyte compounds of low to moderate solute polarity on a silica-based, endcapped, C18 stationary phase with water‒acetonitrile and water–methanol mobile phases whose elution strength was varied via the volumetric solvent ratio. Detailed information about the analyte-specific interfacial dynamics received from molecular dynamics simulations was incorporated through appropriate linker schemes into Brownian dynamics diffusion simulations in three hierarchical porosity models received from physical reconstructions of silica monoliths with a mean macropore size of 1.23 µm and mean mesopore sizes of 12.3, 21.3, or 25.7 nm. The mean mesopore size was found to have a similar influence on the effective mesopore diffusivity as the analyte polarity and the mobile-phase elution strength, which together determine the analyte residence time on a column. A smaller mesopore size attenuated the increase of the effective mesopore diffusivity with increasing mobile-phase elution strength significantly. The effective bed diffusivity was limited by the analyte residence time rather than by morphological details of the mesopore space. The stronger an analyte was retained by the chromatographic interface inside the mesopores, the slower became the mass transfer between the pore space hierarchies and the lower was the effective bed diffusivity. The B-terms of the plate height equation were finally generated with the bed diffusivities and phase-based retention factors derived from the hierarchical porosity models using additional information about the stationary-phase limit obtained from the analysis of analyte–bonded phase contacts. The B-terms highlight analyte- and mobile phase-specific behavior relevant to isocratic and gradient elution conditions in chromatographic practice. In particular, U-shaped B-term curves are observed due to the dominating contribution of the retention factor and the bed diffusivity to the B-term at low and high elution strength of the mobile phase, respectively.

Chromatographic Separation of Hydrophilic Organophosphates on a Porous Graphitized Carbon Sorbent Hypercarb Using an Aqueous Solution of Formic Acid As a Mobile Phase

Chromatographic Separation of Hydrophilic Organophosphates on a Porous Graphitized Carbon Sorbent Hypercarb Using an Aqueous Solution of Formic Acid As a Mobile Phase

Pharmacokinetic Analysis of Gatifloxacin and Dexamethasone in Rabbit Ocular Biofluid Using a Sensitive and Selective LC-MS/MS Method.

Bacterial keratitis (BK) is an infection that causes inflammation of the cornea and, if severe, can result in blindness. Topical fluoroquinolones combined with corticosteroids have been shown to be useful in the treatment of BK. A rapid, selective, and sensitive bioanalytical method for simultaneous quantification of Gatifloxacin (GAT) and Dexamethasone (DEX) has been developed and validated using tandem mass spectrometry (LC-MS/MS). Optimal separation was accomplished in under 5 min using an Agilent Zorbax C18 column (100 mm × 4.6 mm, 3.5 μm). The mobile phase was composed of a blend of 0.2% formic acid in triple distilled water and methanol with a flow rate of 0.65 mL/min in isocratic mode. GAT and DEX were detected in positive electrospray ionization multiple reaction monitoring mode (MRM), and the retention time was found to be at 1.64 and 2.93 min, respectively. The linearity of GAT and DEX was found to be in the range of 1.56-400 ng mL-1 with good precision and accuracy. The method was validated according to USFDA regulatory guidelines. The validated method was effectively utilized for preclinical pharmacokinetic analysis of GAT and DEX in rabbit tear fluid following the topical application of a commercial formulation.