Rapid High-pressure Liquid Research Articles

Increasing yield of in vitro transcription reaction with at-line high pressure liquid chromatography monitoring.

The COVID-19 pandemic triggered an unprecedented rate of development of messenger ribonucleic acid (mRNA) vaccines, which are produced by in vitro transcription reactions. The latter has been the focus of intense development to increase productivity and decrease cost. Optimization of in vitro transcription (IVT) depends on understanding the impact of individual reagents on the kinetics of mRNA production and the consumption of building blocks, which is hampered by slow, low-throughput, end-point analytics. We implemented a workflow based on rapid at-line high pressure liquid chromatography (HPLC) monitoring of consumption of nucleoside triphosphates (NTPs) with concomitant production of mRNA, with a sub-3 min read-out, allowing for adjustment of IVT reaction parameters with minimal time lag. IVT was converted to fed-batch resulting in doubling the reaction yield compared to batch IVT protocol, reaching 10 mg/ml for multiple constructs. When coupled with exonuclease digestion, HPLC analytics for quantification of mRNA was extended to monitoring capping efficiency of produced mRNA. When HPLC monitoring was applied to production of an anti-reverse cap analog (ARCA)-capped mRNA construct, which requires an approximate 4:1 ARCA:guanidine triphosphate ratio, the optimized fed-batch approach achieved productivity of 9 mg/ml with 79% capping. The study provides a methodological platform for optimization of factors influencing IVT reactions, converting the reaction from batch to fed-batch mode, determining reaction kinetics, which are critical for optimization of continuous addition of reagents, thus in principle enabling continuous manufacturing of mRNA.

NEW RP-HPLC METHOD DEVELOPMENT AND VALIDATION FOR THE ESTIMATION OF ANTIFUNGAL DRUG ITRACONAZOLE IN ITRACONAZOLE LOADED INVASOMES GEL

A new simple, accurate, rapid, selective and robust high pressure liquid chromatography (HPLC) method was developed and validated for estimation of itraconazole (ITZ) in bulk and prepared in house invasomes gel formulation. Acetonitrile and Methanol was used as a mobile phase for chromatographic separation in the ratio of 20mM KH2PO4: Acetonitrile (pH adjusted to 4.0 with OPA) at flow rate of 1.0 ml/min and estimation on Thermo C18 (250 × 4.6 mm) in the ratio of 50:50 v/v at flow rate of 1.0 ml/min. The detection was carried out with UV detector set at 262nm. The retention time for itraconazole was found to be 2.258±0.095 minutes. The linearity range for itraconazole was found to be 5-25 μg/ml with coefficient of linear regression 0.998. The method was validated in accordance with the requirements of International Conference on Harmonization (ICHQ2 A 8 B) guidelines for specificity, linearity, accuracy, precision, LOD 8 LOQ.

Method development and validation for simultaneous estimation of amlodipine besylate and enalapril maleate in solid dosage form

A rapid, sensitive, specific, accurate and precise high pressure liquid chromatographic method (HPLC) method involving UV detection has been developed for the determination and quantification of Amlodipine Besylate and Enalapril maleate in bulk and combined dosage form. The determination was carried out on a Phenomenex C18 column (Dimention : 250 x 4.6 mm, 5 μm). The sample was analysed using filtered and degassed mixture of methanol : 0.1N HCl (1:1) as mobile phase at a flow rate of 1ml/min and effluent was monitored at 218nm. The retention time for Amlodipine besylate was 7.6 min and for Enalapril maleate 3.2 min. Amlodipine besylate and Enalapril maleate showed a linear response in the concentration range of 10-50μg/ml. The correlation co-efficient ('r' value) for Amlodipine besylate and Enalapril maleate was 0.9992 and 0.9994, respectively. The method was validated in terms of linearity, precision, accuracy, specificity, robustness and solution stability. The proposed method can be used for routine analysis of Amlodipine Besylate and Enalapril maleate in bulk and combined dosage form

A rapid LC-MS/MS method for simultaneous determination of nicotine and its key derivatives including hydroxylation isomers

A rapid high pressure liquid chromatography coupled with tandem mass spectrometry method (LC-MS/MS) was developed for simultaneously determining nicotine (NT) and its derivatives including cotinine (CT), trans-3-hydroxycotinine (HC), nornicotine (NNT), anatabine (AT), anabasine (AB) and amino ketone (4-(methylamino)-1-(3-pyridyl)1-butanone) (AK) as well as two important metabolites isomers 2-hydroxynicotine (2-HN) and 6-hydroxynicotine (6-HN) in tobacco leaves. Different from other methods, 2-HN and 6-HN isomers can be successfully separated and quantified through a combination of amide and C18 columns and optimized multiple reaction monitoring mode of tandem mass spectrometry. The limits of detections (LOD) and quantification (LOQ) of these compounds were 0.02 and 0.06 ng/mL for CT and HC, 0.05 and 0.15 ng/mL for AK and 6-HN, and 0.1 and 0.3 ng/mL for AB, NNT, AT, 2-HN and NT, respectively. Their recoveries ranged from 86.1% for 2-HN to 102.2% for AK by using solvent extraction assisted with ultrasonication. The developed method was validated through determination of the compounds in different regular and genetically modified (for lowering nicotine) tobacco leaves. Conclusively, it is a robust and rapid method for simultaneous quantification of nicotine and its derivatives in tobacco products. The protocol of the method could also be applied in developing analysis method for tracking the toxic substances synthesis and metabolism in tobacco leaves or other herbal plants.

HPLC–MS/MS method for the simultaneous quantification of desmethylmebeverine acid, mebeverine acid and mebeverine alcohol in human plasma along with its application to a pharmacokinetics study

A new simple, rapid and sensitive high pressure liquid chromatography-tandem mass spectrometry (HPLC–MS/MS) method was developed and validated for simultaneous analysis of mebeverine metabolites as: mebeverine alcohol (MAL), mebeverine acid (MAC) and desmethylmebeverine acid (DMAC) in human plasma. Sample preparation was performed by protein precipitation following the separation of analytes using an Acquity UPLC BEN C8 column 1.7 mm 2.1×50mm (Waters, USA). 2H5-desmethylmebeverine acid (2H5-DMAC) was used as the internal standard (IS). The proposed method was validated with linear ranges of 0.1–10ng/mL; 1–100ng/mL and 5–1000ng/mL for MAL, MAC and DMAC, respectively. Accuracy for all analytes (%RE), given as deviation between nominal and measured concentration and assay variability (CV) ranged from −4.04% to 4.60% and from 0.31% to 6.43% respectively both for within- and between-run. The overall recoveries for all metabolites were above 85%. The proposed method was used successfully for analysis of real samples from a pharmacokinetics study.

Simultaneous quantification of 5 main components of Psoralea corylifolia L. in rats' plasma by utilizing ultra high pressure liquid chromatography tandem mass spectrometry.

Psoralea corylifolia L. has long been used in traditional Chinese medicine for treating and preventing many diseases. A group of flavonoid components are regarded as the active principals within the seeds. In this research, a rapid, accurate and sensitive ultra high pressure liquid chromatography tandem mass spectrometry (UHPLC/MS/MS) method has been established for simultaneous quantification of its 5 main components, namely, neobavaisoflavone, bavachin, isobavachalcone, bavachinin and corylifol A in rats' plasma after the rats were orally administrated with Buguzhi extract. Negative ion electrospray mode was applied in the detection process. Multiple reactions monitoring (MRM) mode was utilized for simultaneous quantitative analyzing of neobavaisoflavone (m/z 321.1→m/z 265.1), bavachin (m/z 323.1→m/z 119.0), isobavachalcone (m/z 323.2→m/z 119.0), bavachinin (m/z 337.2→m/z 119.0), corylifol A (m/z 389.2→m/z 277.0) and liquiritigenin (Internal Standard, m/z 255.1→m/z 119.0). Chromatographic separation of the above mentioned components was conducted on a Waters BEH-C18 column (100 mm×2.1mm, 1.7μm) with gradient elution system at flow rate of 0.3mL/min. The mobile phase was composed of acetonitrile and 0.1% formic acid solution. The lower limit of quantification (LLOQ) for each of the above analytes was 0.5ng/mL. Each of the analytes exhibited good linearity within the concentration range of 0.5-100ng/mL. The method was fully validated for its selectivity, accuracy, precision, stability, matrix effect and extraction recovery. The validated method has been successfully applied for simultaneous determination of the 5 flavonoids in rat plasma for the first time.

Simple and Rapid High Pressure Liquid Chromatography Method for Estimation of Ethionamide in Plasma

A high performance liquid chromatographic method for determination of Ethionamide (ETH) in human plasma was developed. The method involved deproteinisation of the sample with perchloric acid and analysis of the supernatant using a reversed-phase CN column (150mm) and a wavelength of 267 nm. The mobile phase consisted of Milli-Q water and Methanol (85:15, v/v) containing 0.05% Perchloric acid and 0.1% tetrabutyl N-ammonium hydroxide. The assay was specific for ETH and linear from 0.25 to 10.0μg/ml. The relative standard deviation of intra- and inter-day assays was lower than 10%. The average recovery of ETH from plasma was 91%. Due to its simplicity, the assay can be used for pharmacokinetic studies of ETH and to check patient adherence to tuberculosis treatment.

Development of a Simple Chromatographic Method for the Determination of Piracetam in Human Plasma and Its Pharmacokinetic Evaluation

The objective of study was to develop an accurate and reproducible HPLC method for determination of piracetam in human plasma and to evaluate pharmacokinetic parameters of 800 mg piracetam. A simple, rapid, accurate, precise and sensitive high pressure liquid chromatography method has been developed and subsequently validated for determination of piracetam. This study represents the results of a randomized, single-dose and single-period in 18 healthy male volunteers to assess pharmacokinetic parameters of 800 mg piracetam tablets. Various pharmacokinetic parameters were determined from plasma for piracetam and found to be in good agreement with previous reported values. The data was analyzed by using Kinetica® version 4.4 according to non-compartment model of pharmacokinetic analysis and after comparison with previous studies, no significant differences were found in present study of tested product. The major pharmacokinetic parameters for piracetam were as follows: t1/2 was (4.40 ± 0.179) h; Tmax value was (2.33 ± 0.105) h; Cmax was (14.53 ± 0.282) µg/mL; the AUC(0-∞) was (59.19 ± 4.402) µg · h/mL. AUMC(0-∞) was (367.23 ± 38.96) µg. (h)(2)/mL; Ke was (0.16 ± 0.006) h; MRT was (5.80 ± 0.227) h; Vd was (96.36 ± 8.917 L). A rapid, accurate and precise high pressure liquid chromatography method was developed and validated before the study. It is concluded that this method is very useful for the analysis of pharmacokinetic parameters, in human plasma and assured the safety and efficacy of piracetam, can be effectively used in medical practice.

Effect of strain and cultural conditions on the production of cytochalasin B by the potential mycoherbicide Pyrenophora semeniperda (Pleosporaceae, Pleosporales)

The seed pathogen Pyrenophora semeniperda has demonstrated potential as a mycoherbicidal biocontrol for eliminating persistent seed banks of annual bromes on western North American rangelands. This pathogen exhibits variation in virulence that is related to mycelial growth rate, but direct laboratory tests of virulence on seeds often have low repeatability. We developed a rapid and sensitive high pressure liquid chromatography method for quantification of the phytotoxin cytochalasin B in complex mixtures in order to evaluate its use in virulence screening. All 10 strains tested produced large quantities of this metabolite in solid wheat seed culture, with production varying over a fourfold range (535–2256 mg kg−1). No cytochalasin B was produced in liquid potato dextrose broth culture, showing that its synthesis is strongly dependent on cultural conditions. In a Bromus tectorum coleoptile bioassay, solid culture extracts showed mild toxicity similar to the cytochalasin B standard at a concentration equivalent to 10−4 M cytochalasin B (72–95% of control), whereas at 10−3 M equivalent, the extracts exhibited significantly higher toxicity (8–18% of control) than the cytochalasin B standard (34% of control). This suggests the possible presence of other phytotoxic metabolites. Cytochalasin B production in solid wheat seed culture exhibited the predicted significant negative correlation with mycelial growth rate on potato dextrose agar, but the correlation was not very strong, possibly because cytochalasin B production and growth rate were measured under different cultural conditions.

A rapid method for the determination of artemisinin and its biosynthetic precursors in Artemisia annua L. crude extracts

A rapid high-pressure liquid chromatography (HPLC) tandem mass spectrometry (TQD) method for the determination of artemisinin, 9-epi-artemisinin, artemisitene, dihydroartemisinic acid, artemisinic acid and arteannuin B in Artemisia annua extracts is described. Detection and quantification of 9-epi-artemisinin in crude extracts are reported for the first time. In this method all six metabolites are resolved and eluted within 6min with minimal sample preparation. A recovery of between 96.25% and 103.59% was obtained for all metabolites analysed and the standard curves were linear (r2>0.99) over the concentration range of 0.15–10μgmL−1 for artemisinin, 9-epi-artemisinin, artemisitene and arteannuin B, and the range of 3.75–120μgmL−1 for dihydroartemisinic acid and artemisinic acid. All validation indices were satisfactory, showing the method to be robust, quick, sensitive and adequate for a range of applications including high throughput (HTP) analysis.

Development and validation of a rapid ultra high pressure liquid chromatographic method for the determination of methylxanthines in herbal infusions

An ultra high pressure liquid chromatographic method coupled with diode array detector (UHPLC-DAD) has been developed and validated for the fast separation and determination of three major methylxanthines, i.e., caffeine, theophylline and theobromine, in various herbal beverages. Isocratic elution using 0.1vol% formic acid/CH3OH (92.5:7.5, v/v) enabled the completion of the separation cycle in less than 3min using a flow rate of 0.7mL/min and a column temperature of 50°C. Validation of the method included linearity (0.5-50mg/L), limits of detection (12-35μg/L) and quantification (40-120μg/L), precision, matrix effect and accuracy. The percent recoveries ranged between 90 and 108%.

SELECTIVE REVERSED PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY FOR THE SIMULTANEOUS DETERMINATION OF SOME PHARMACEUTICAL BINARY MIXTURES CONTAINING NSAIDS

A simple, rapid, sensitive, and economic reversed-phase High Pressure Liquid Chromatography method was developed for the simultaneous evaluation of five binary mixtures in their pharmaceutical preparations. The investigated mixtures were Ibuprofen(IP)-paracetamol(PC), Ibuprofen(IP)-Chlorzoxazone(CZ), Ibuprofen(IP)- Methocarbamol(MC), Ketoprofen(KP)-Chlorzoxazone(CZ), and Diclofenac sodium(DS)-Lidocaine hydrochloride(LC). The chromatographic separation was performed using isochratic mode at a flow rate 1 mL/ min, ODS C18 column, and UV detection at 254 nm. The mobile phase consisted of acetonitrile:phosphate buffer, pH 5 (60:40). The parameters affecting separation of the investigated drugs were studied. Under the optimum assay conditions, the method gave linear calibration curves with good correlation coefficients. The limits of detection (LOD) and limits of quantitation (LOQ) ranged from 0.34–5.33 µg/mL and 1.03–16 µg/mL, respectively, indicating high sensitivity of the method. The assay of the investigated binary mixtures is completed in less than 5 min. The proposed method was successfully applied to the simultaneous analysis of the drugs in their pharmaceutical binary mixtures in dosage forms with good recoveries and no significant differences between proposed and reported method. This method can be routinely applied to the quality control of the investigated binary mixtures in their pharmaceutical formulations.

Detection and quantification of tricyclic antidepressants and other psychoactive drugs in urine by HPLC/MS/MS for pain management compliance testing.

A sensitive, specific, and rapid high-pressure liquid chromatography/mass spectrometry/mass spectrometry method was developed for the quantitation of 11 tricyclic antidepressants and/or their metabolites; fluoxetine and norfluoxetine; cyclobenzaprine; and trazodone in urine. Samples were alkalinized with 0.2 N NaOH and extracted into 2 ml of hexane: ethyl acetate (1:1), evaporated to dryness, and reconstituted with 100 μl of 20 mM ammonium formate: methanol (20:80). The chromatographic separation was performed using an Allure Biphenyl 100 × 3.2 mm, 5-μ column with a mobile phase consisting of 20 mM ammonium formate: methanol (20:80 v/v) at a flow rate of 0.5 ml/min. The detection was accomplished by multiple-reaction monitoring via electrospray ionization source operating in the positive ionization mode. The calibration curve was linear over the investigated concentration range, 25-2,000 ng/ml, for each analyte using 1.0 ml of urine. The lower limit of quantitation for each analyte was 25 ng/ml. The intra- and inter-day precisions had coefficient of variation less than 15% and the accuracy was within the range from 88% to 109%. The method proved adequate for the tricyclic antidepressants analysis of urine for emergency clinical toxicology and pain management compliance testing.

Ceftazidime determination in serum by high-pressure liquid chromatography.

A rapid and sensitive high-pressure liquid chromatographic method with simple sample preparation was developed for the quantitative analysis of the beta-lactam antibiotic ceftazidime (CAS 78439-06-2, Fortum). A good linear relationship was established between the peak area and the amount of ceftazidime injected over a concentration range of 1 to 200 microg/ml. The detection limit of the method was calculated to be 0.9 microg/ml. Stability was shown at 4 degrees C and at -196 degrees C for time periods of 2 h and 84 days, respectively.

A HPLC-MS method to detect and quantify guanfacine in urine

Guanfacine, an α2-adrenergic α2A)agonist long indicated to treat hypertension, is now being used to treat attention deficit-hyperactivity disorder (ADHD) in adolescents. This new therapeutic use may require urine testing to document compliance or abuse. A simple rapid high pressure liquid chromatography-mass spectrometry (HPLC-MS) method to detect and quantify guanfacine in urine following therapeutic administration is presented. Guanfacine and protriptyline internal standard were extracted from alkalinized urine with ethyl acetate. The organic layer was evaporated, reconstituted with mobile phase and analyzed on a YMC Basic S-5 micron, 2.0 × 150 mm HPLC column connected to an MS detector operated in positive electrospray ionization mode with selected ion resonance. Elution times were <5 min. The analytical measurement range for guanfacine was 20-2000 ng/mL. The limit of detection and quantitation were 5 ng/mL and 20 ng/mL, respectively. Precision as %CV was <15% at 40, 100 and 500 ng/mL (n=6). Percentage recovery using the same concentrations was >89%. Interference with drugs and biological constituents was assessed; no interferences were noted. Analysis of 100 random post-diagnostic urine specimens yielded 11 guanfacine positive results with concentrations ranging from 11 to 6390 ng/mL. This HPLC-MS method provides a simple and rapid method for the routine detection and quantitation of guanfacine in urine specimens.

A Rapid and Selective LC‐MS/MS Method for Quantification of Quetiapine in Human Plasma and its Application to Pharmacokinetic Study on Indian Schizophrenia Patients

A rapid, robust and selective high pressure liquid chromatography–positive electrospray ionization tandem mass spectrometry method has been developed and validated for the quantification of quetiapine (QUE) in human plasma with K2EDTA using oxcarbazepine (IS) as an internal standard. Analyte and internal standard were extracted from human plasma by solid‐phase extraction using acetonitrile. The eluted samples were chromatographed on a C18 column by using a 10:75:15v/v mixture of ammonium formate buffer (5 mM, pH 4.50) and acetonitrile and methanol as an isocratic mobile phase at a flow rate of 0.4 mL/min and analyzed by mass spectrometry in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 384.3/253.2 for Quetiapine and m/z 253.1/208.1 for the internal standard. The assay exhibited a linear dynamic range of 5.01 ‐ 2501.04 ng/mL for quetiapine in human plasma. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.5 min for each sample made it possible to analyze 300 patient plasma samples per day. The validated method has been successfully used for the estimation of quetiapine in real time schizophrenia patient’s plasma samples for pharmacokinetic study.

Development and Validation of RP-HPLC Method for The Simultaneous Estimation of Amoxicillin Trihydrate and Bromhexine Hydrochloride from Oily Suspension

A new simple, rapid and precise reverse phase high pressure liquid chromatography (RP-HPLC) method was developed for the simultaneous estimation of amoxicillin trihydrate and bromhexine hydrochloride from oily suspension. An ODS C 18 (250 X 4.5mm ID), 5 μ particle size with mobile phase methanol and glacial acetic acid (50:50 v/v) were used. The flow rate was 1.0ml/min and responses were measured at 254 nm. The retention time for amoxicillin trihydrate and bromhexine hydrochloride were observed at 3.04 and 8.18 min. respectively. Linearity for amoxicillin trihydrate and bromhexine hydrochloride were in the range of 8-50 mcg/ml and 5-25 mcg/mL respectively. Percent recovery was 99.54% and 98.65% for amoxicillin trihydrate and bromhexine hydrochloride respectively. The proposed method can be applied for the routine analysis of amoxicillin trihydrate and bromhexine hydrochloride in combination.

A Rapid Determination of Cinnarizine in Bulk and Pharmaceutical Dosage Form by LC

A simple, selective, rapid and precise reverse phase high pressure liquid chromatographic method has been developed for the estimation of cinnarizine from pharmaceutical formulation. The method was developed using MICRA‐NPS C18 (length×OD×ID =33×8.0×6.0 mm, 1.5 μm) column with a mobile phase consisting of acetonitrile, triethylamine buffer (adjusted to pH 4.5 with 10% w/v potassium hydroxide) and tetrahydrofuran in the ratio 30:66:4 respectively, at a flow rate of 0.5 mL/min. Wavelength was fixed at 253 nm. The developed method was validated for linearity, accuracy, precision, limit of detection and limit of quantitation. The proposed method can be used for the routine estimation of cinnarizine in pharmaceutical dosage form.

Fast HPLC–ECD analysis of ascorbic acid, dehydroascorbic acid and uric acid

A robust and rapid high-pressure liquid chromatography–electrochemical detection (HPLC–ECD) method was developed and validated for the accurate determination of ascorbic acid (AA) and uric acid (UA), in human plasma. Dehydroascorbic acid (DHAA) was indirectly measured by subtracting native ascorbic acid from total ascorbic acid concentrations; the latter was obtained after chemical reduction. A stable electrochemical active internal standard (homogentisic acid) was added for the accurate quantification of the analytes. The analyses were performed on a reverse-phase column with traditional HPLC and ultra-HPLC (UHPLC). The UHPLC method showed increased sensitivity with detection limit of 0.05 ng for both AA and UA, 2 times lower compared to conventional HPLC. UHPLC also reduced run times fourfold with less waste generation. Both assays showed good accuracy and precision, the intra- and inter-day CVs of AA and UA analysis are less than 7%.

A validated RP-HPLC method for simultaneous estimation of nebivolol and hydrochlorothiazide in tablets

A simple, selective, rapid, precise and economical reverse phase high pressure liquid chromatographic method has been developed for the simultaneous estimation of nebivolol and hydrochlorthiazide from pharmaceutical formulation. Phenomenex Gemini C18 (25 cm×4.6 mm i.d., 5 μ) column with a mobile phase consisting of acetonitrile: 50mM ammonium acetate (adjusted to pH 3.5 using orthophosphoric acid) (70:30 v/v) at a flow rate of 1.0 ml/min was used. Detection was carried out at 254 nm. Probenecid was used as an internal standard. The retention times of probenecid, nebivolol and hydrochlorthiazide were 13.05, 3.32 and 4.25 min, respectively. The developed method was validated in terms of accuracy, precision, linearity, limit of detection, limit of quantitation and solution stability. The proposed method can be used for the estimation of these drugs in combined dosage forms.