Isocratic Mobile Phase System Research Articles

QbD green analytical procedure for the quantification of tolvaptan by utilizing stability indicating UHPLC method

For the first time a new QbD-assisted green stability indicating ultra-high-performance liquid chromatography (UHPLC) method was developed and validated for quantifying Tolvaptan. The method is simple, quick, cost-effective, and stable, and it was used to formulate a quality target product profile (QTPP) with strategically defined critical analytical attributes (CAAs) to meet specific criteria. Chromatographic separation was undertaken using a 10 cm long column of ACE excel super C18 with an interior diameter of 2.1 mm and particle size of 1.7 µm. The analysis was performed under controlled conditions at 25 ℃ with the mobile phase flowing at a rate of 0.2 mL/min and detection occurring at 220 nm. Injected 3 µL of standard by using an isocratic mobile phase system consisting of acetonitrile and water in a 95:5 v/v ratio. The diluents, prepared by mixing acetonitrile with water at a 90:10 volumetric ratio, were utilized. The analyte’s retention time was determined to be 1.63 min. The developed method provided reliable results with accuracy exceeding 99% and a correlation coefficient exceeding 0.999 ranged between 10 and 150 µg/mL across the range for LOQ—150% levels. Notably, during forced degradation testing, Tolvaptan exhibited susceptibility to acidic hydrolysis. The method effectively separated degradation products during stress testing, demonstrating its stability-indicating status. Environmental sustainability assessment of the developed method was conducted through the investigation of various indicators of Complex GAPI, Analytical Eco scale and Analytical GREEness and it was concluded the optimized method aligns with environmentally friendly practices.

Unveiling Pharmacokinetics and Drug Interaction of Linagliptin and Pioglitazone HCl in Rat Plasma Using LC-MS/MS.

The linagliptin (LIN) and pioglitazone HCl (PIO) combination, currently undergoing phase III clinical trials for diabetes mellitus treatment, demonstrated significant improvements in glycemic control. However, the absence of an analytical method for simultaneous determination in biological fluids highlights a crucial gap. This underscores the pressing need for sensitive bioanalytical methods, emphasizing the paramount importance of developing such tools to advance diabetes management strategies and enhance patient care. Herein, a sensitive reverse-phase high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry method was developed for simultaneous determination of LIN and PIO in rat plasma using alogliptin as an internal standard. Chromatographic separation was performed on an Agilent Eclipse Plus C18 (4.6 × 100 mm, 3.5 μm) using an isocratic mobile phase system consisting of ammonium formate (pH 4.5) and methanol using an acetonitrile-induced protein precipitation technique for sample preparation. Multiple reaction monitoring in positive ion mode was used for quantitation of the precursor to production at m/z 473.2 → 419.9 for LIN, 357.1 → 134.2 for PIO, and 340.3 → 116.1 for ALO. The linearity range was 0.5 to 100 and 1 to 2000 ng/mL for LIN and PIO, respectively. The developed method was validated as per US-FDA guidelines and successfully applied to clinical pharmacokinetic and drug-drug interaction studies with a single oral administration of LIN and PIO in rat plasma. Pharmacokinetic parameters of LIN were significantly influenced by the concomitant administration of PIO and vice versa. Molecular modeling revealed the significant interaction of LIN and PIO with P-glycoprotein. Therefore, the drug-drug interaction between LIN and PIO deserves further study to improve drug therapy and prevent dangerous adverse effects.

Analytical quality by design approach to develop an eco-friendly RP-HPLC method for estimation of irbesartan in chitosan polymeric nanoparticles: forced degradation studies and assessment of in vitro release mathematical modelling.

Irbesartan is an angiotensin converting enzyme blocker, primarily utilized for the management of hypertension and the mitigation of diabetic nephropathy progression. The present study introduces rapid, robust and environmentally sustainable reverse phase high performance liquid chromatography (RP-HPLC) validated under the analytical quality by design (AQbD) framework according to ICH guidelines. Utilizing a central composite design, the method's systemic optimization was achieved, ensuring reproducibility and accuracy. Chromatographic separation was accomplished utilizing an ethanol and sodium acetate buffer (60 : 40 v/v) isocratic mobile phase system on a zorbax sb C18 column, with a flow rate of at 0.6 mL min-1. Studies on forced degradation outlined stability of irbesartan and its degradation processes, enhancing our understanding of its chemical robustness under varied conditions. Complementing the green chemistry paradigm, the method's environmental impact was critically assessed, affirming its alignment with sustainability objectives. The validated method proved pivotal in determining the percent entrapment and loading efficiency of the formulated nanoparticles and holds potential for application in biological matrices. Furthermore, the encapsulation of IRB within chitosan nanoparticles was explored to assess release kinetics and enhance bioavailability. This study not only advances the analytical sciences by merging eco-friendly practices with method development but also broadens the applicative landscape of HPLC methodologies in drug delivery research.

Development of a Fast and Sensitive UPLC–MS/MS Analytical Methodology for Fenebrutinib Estimation in Human Liver Microsomes: In Vitro and In Silico Metabolic Stability Evaluation

Fenebrutinib (GDC-0853; FNB) is an oral small molecule that was developed by Roche Pharmaceuticals to slow multiple sclerosis progression. FNB is a reversible bruton tyrosine kinase (BTK) inhibitor, which showed the maximum potency of BTK inhibitors in phase III clinical trials for multiple sclerosis. In the current study, a fast, specific, and sensitive UPLC-MS/MS method for FNB quantification in human liver microsomes (HLMs) was established with application to the evaluation of metabolic stability. The UPLC-MS/MS methodology was verified using the stated USFDA validation guidelines for bioanalytical methodologies that involve selectivity, linearity, accuracy and precision, carryover and extraction recovery, stability, and matrix effect. The FNB calibration curve displayed a linearity in the range from 1 ng/mL to 3000 ng/mL (y = 1.731x + 2.013; R2: 0.9954; RSD < 4.37%) in the HLMs matrix. The limit of quantification was 0.88 ng/mL, which verified the UPLC-MS/MS analytical method sensitivity. The intraday and interday precision and accuracy results of the developed UPLC-MS/MS method were −3.99–14.0% and 0.52–3.83%, respectively. FNB and savolitinib (SVB) (internal standard) were chromatographically separated utilizing an isocratic mobile phase system with a ZORBAX Eclipse plus-C18 (50 mm, 2.1 mm, and 1.8 μm) column. The metabolic stability parameters for FNB, involving high intrinsic clearance (58.21 mL/min/kg) and a short in vitro half-life (13.93 min), revealed the high extraction ratio of FNB. Reviewing the literature revealed that the current UPLC-MS/MS method is the first analytical method for FNB quantification in the HLMs matrix with application to the assessment of FNB metabolic stability.

Development of an LC-MS/MS Method for Quantification of Sapitinib in Human Liver Microsomes: In Silico and In Vitro Metabolic Stability Evaluation.

Sapitinib (AZD8931, SPT) is a tyrosine kinase inhibitor of the epidermal growth factor receptor (EGFR) family (pan-erbB). In multiple tumor cell lines, STP has been shown to be a much more potent inhibitor of EGF-driven cellular proliferation than gefitinib. In the current study, a highly sensitive, rapid, and specific LC-MS/MS analytical method for the estimation of SPT in human liver microsomes (HLMs) was established with application to metabolic stability assessment. The LC-MS/MS analytical method was validated in terms of linearity, selectivity, precision, accuracy, matrix effect, extraction recovery, carryover, and stability following the FDA guidelines for bioanalytical method validation. SPT was detected using electrospray ionization (ESI) as an ionization source under multiple reaction monitoring (MRM) in the positive ion mode. The IS-normalized matrix factor and extraction recovery were acceptable for the bioanalysis of SPT. The SPT calibration curve was linear, from 1 ng/mL to 3000 ng/mL HLM matrix samples, with a linear regression equation of y = 1.7298x + 3.62941 (r2 = 0.9949). The intraday and interday accuracy and precision values of the LC-MS/MS method were -1.45-7.25% and 0.29-6.31%, respectively. SPT and filgotinib (FGT) (internal standard; IS) were separated through the use of an isocratic mobile phase system with a Luna 3 µm PFP(2) column (150 × 4.6 mm) stationary phase column. The limit of quantification (LOQ) was 0.88 ng/mL, confirming the LC-MS/MS method sensitivity. The intrinsic clearance and in vitro half-life of STP were 38.48 mL/min/kg and 21.07 min, respectively. STP exhibited a moderate extraction ratio that revealed good bioavailability. The literature review demonstrated that the current analytical method is the first developed LC-MS/MS method for the quantification of SPT in an HLM matrix with application to SPT metabolic stability evaluation.

Estimation of zorifertinib metabolic stability in human liver microsomes using LC–MS/MS

Zorifertinib (AZD-3759; ZFB) is a novel, potent, oral, small molecule used to treat non-small cell lung cancer. ZFB is an epidermal growth factor receptor inhibitor that is capable of crossing blood–brain barrier. The in silico metabolic software used for ZFB metabolic stability prediction was the StarDrop software package (WhichP450 module). An LC-MS/MS analytical method (fast and accurate) was established for ZFB quantification in human liver microsomes (HLMs) in order to estimate its metabolic stability. ZFB and encorafenib (ENF) (internal standard; IS) were separated through the use of an isocratic mobile phase system with a C8 stationary phase column. The LC-MS/MS method for ZFB exhibited linearity in the range of 5 ng/mL to 500 ng/mL in HLMs matrix with a linear regression equation: y = 0.2438x - 0.341 (R² = 0.9992). The limit of quantification (LOQ) was 3.78 ng/mL confirming the LC-MS/MS method sensitivity. The inter- and intraday accuracy and precision were less than 9.56% confirming the reproducibility of the LC-MS/MS method. The intrinsic clearance and in vitro half-life of ZFB were 32.5 µL/min/mg and 21.33 min, respectively. ZFB exhibited a moderate extraction ratio that revealed good bioavailability. Literature review demonstrated that the developed analytical method is the first developed LC-MS/MS method for determining ZFB metabolic stability.

Reversed-Phase Liquid Chromatographic Internal Standard Method Using Losartan Potassium for Quantitative Estimation of Ivabradine Hydrochloride in Pharmaceutical Tablet Dosage Form and Plasma

A reversed-phase liquid chromatographic method for ivabradine hydrochloride using Diode Array Detector (DAD) and internal standard technique was developed and validated according to ICH and SWGTOX guidelines. The prime objective of this study was to develop a precise and accurate method that can be equally applicable to biological (plasma) as well as non-biological (active pharmaceutical ingredient and pharmaceutical tablets) matrices. Losartan potassium was used as an internal standard due to its easy availability. After liquid-liquid extraction using acetonitrile, the ivabradine hydrochloride and internal standard were chromatographed on Agilent 1200 series HPLC system equipped with DAD detector, auto-sampler and chemstation software. Analytical separation was achieved on Agilent C-18 (5µm, 25cm x 4.6mm) reversed-phase column at 30°C column oven temperature, 10μL injection volume and 286nm wavelength. Isocratic mobile phase system comprised of 60:40 v/v ratio of HPLC grade methanol and water adjusted to pH 6.8 using orthophosphoric acid was employed with 1mL/min flow rate. The method linear range was 0.025-3µg/mL (25-3000ng/mL) for pharmaceutical tablets and plasma with the coefficient of linearity ranged 0.997-0.999. Results for precision, accuracy, recovery, stability and matrix effect studies were within acceptable limits for both plasma and tablets. Method was successfully applied to the commercial tablet products and patient plasma samples to estimate the amount of ivabradine hydrochloride.

In Vitro Culture of Rosmarinus officinalis L. in a Temporary Immersion System: Influence of Two Phytohormones on Plant Growth and Carnosol Production.

Emerging infectious diseases have become a major global problem with public health and economic consequences. It is an urgent need to develop new anti-infective therapies. The natural diterpene carnosol exhibit a wide variety of interesting antibacterial and antiviral properties, and it is considered a theoretical inhibitor of COVID-19 Mpro. However, this compound is present in the family Lamiaceae in low quantities. To obtain carnosol in concentrations high enough to develop pharmacological studies, we evaluated the efficiency of a micropropagation protocol of Rosmarinus officinalis using a solid medium and a temporary immersion system (TIS), as well as the effect of 6-benzylaminopurine (6-BAP) and α-naphthaleneacetic acid (NAA) on the growth of shoots. Moreover, we developed and validated an analytical method to quantify carnosol using the H-point standard additions method in the high-performance liquid chromatography diode array detector (HPLC-DAD). After 30 days of culture, TIS produced the maximum number of shoots per explant (24.33 ± 1.15) on a liquid medium supplemented with 6-BAP at 5.0 mg L−1. Next, we also evaluated the effect of immersion time and frequency for TIS. After 72 days of culture, the best results were obtained with an immersion cycle of 1 min every 12 h, yielding 170.33 ± 29.40 shoots. The quantification of carnosol on the samples was performed at a flow rate of 1.2 mL min−1 using binary isocratic mobile phase system 60:40 (v/v) 10 mM formic acid (pH 3.0) (A) and acetonitrile (B) on a reverse-phase column. The content of carnosol in the in vitro cultures was around 8-fold higher than in the wild plant. The present study represents an efficient alternative method to obtain carnosol for its pre-clinical and clinical development.

Study of forced-acid/heat degradation and degradant/impurity profile of phenazopyridine hydrochloride through HPLC and spectrofluorimetric analyses

The article handles two related subjects. First, it's the study of forced-acid/heat degradation of phenazopyridine HCl (PAP). The reaction pathway was evidenced with different criteria. A three wavelengths-based reversed phase HPLC method with an isocratic mobile phase system (acetonitrile:acetate buffer of pH 4, 1:1, v/v) was developed as a stability-indicating assay of particular application for simultaneous determination of PAP and its related degradation products/impurity. The analysis time was less than 8 min at a flow rate of 1 mL min-1. The detection and quantification limits of degradation products/impurity were in the ranges of 0.02-0.1 and 0.06-0.3 μg mL-1, respectively. The impurity profile of PAP in drug substance and tablets was investigated and could be determined down to a level between 0.03% and 0.005%. The second subject deals with establishment of spectrofluorimetric method for quantification of phenol (PH) and 2,6-diaminopyridine (DAP), as degradant/impurity in PAP, at trace concentrations. The method is based on measurement of the native fluorescence in acidic ethanol solution (DAP) or in aqueous acidic solution (PH). The method was applied to check the purity/stability of PAP in drug substance and in tablets dosage form.

Determination of Pyridaphenthion in Aqueous and Food Samples by Reverse Phase High Performance Liquid Chromatography (HPLC) after QuEChERS Extraction and Degradation Studies under Ultraviolet (UV) Radiation

This study employed QuEChERS as an efficient sample clean-up and extraction method for the determination of pyridaphenthion in aqueous, fruit and vegetable samples by high performance liquid chromatography (HPLC) coupled with ultraviolet (UV) detection. All of the instrumental parameters were carefully optimized to obtain a sharp analyte signal eluted at an short retention time. The analyte was eluted on a C18 column using an isocratic mobile phase system that was composed of 40:60 (v/v) water:acetonitrile. The linear working range of pyridaphenthion was from 10 μg/L to 50 mg/L, and the respective limits of detection and quantification were determined to be 2.4 and 8.1 μg/L. The instrumental precision was very good with a relative standard deviation value of 1.8%. Spike recovery studies were used to validate the accuracy and applicability of the method for food and aqueous samples. Degradation studies for pyridaphenthion under UV radiation were also performed with a liquid chromatography quadrupole time of flight mass spectrometer.

A micellar HPLC method for simultaneous determination of methocarbamol in three different tablets using single run; application to human plasma and evaluation of the method greenness

Methocarbamol is a well-known centrally acting skeletal muscle relaxant and is usually combined with analgesics like paracetamol, diclofenac and ibuprofen for treatment of musculoskeletal pain. A new approach based on applying green analytical procedures was proposed using micellar chromatographic method for the quantification of methocarbamol in three different combined dosage forms with isocratic mobile phase system in a single run in about 11 min. The separation was performed on C8 column (250 mm × 4.6 mm i.d., 5-μm particle size) with micellar mobile phase consisting of (0.12 M) sodium dodecyl sulfate, (15%) n-propanol, 0.02 M orthophosphoric acid, 0.3% triethylamine, adjusted to pH 6. The eluents were monitored by UV detection at 220 nm. The linearity of the method was acceptable over the concentration ranges of 5–200 μg/mL for methocarbamol, 5–80 μg/mL for paracetamol and 5–100 μg/mL for both diclofenac and ibuprofen. Also, the method was extended to determine methocarbamol in spiked human plasma. The validation of the developed method was evaluated based on ICH guidelines acceptance criteria for linearity, accuracy, precision and robustness. Moreover; the greenness profile of the proposed method was assessed and compared with the published HPLC methods using the analytical Eco-scale as an assessment tool.

A versatile HPLC method with an isocratic single mobile phase system for simultaneous determination of anti-glaucoma formulations containing timolol

Timolol is a non-cardioselective beta blocker and has different combined ophthalmic dosage forms for treatment of glaucoma. This research introduce an HPLC method for the separation of three drugs used in combination with timolol simultaneously by applying isocratic mobile phase system in a single run and the same detection wavelength with short time. The drugs included in the separation procedures are; dorzolamide, brinzolamide, and brimonidine. The HPLC method was carried out through a single mobile phase system, which contains acetonitrile: 0.05M phosphate buffer at the ratio of 30:70, respectively at pH 3.5 and wavelength of 220nm. The method, regarding its simplicity allows determination of the studied drugs simultaneously using single run in about 8minutes. The method was rectilinear in the ranges of concentration: 1.25-25μg/mL for timolol, 4-80μg/mL for dorzolamide, 5-50μg/mL for brinzolamide and 2-20μg/mL for brimonidine. Different factors affecting the separation are thoroughly studied. The developed method was validated based on the official guidelines and the results were compared statistically with previously published methods and showed non-significant difference.

Pharmacokinetics of Quercetin-Loaded Methoxy Poly(ethylene glycol)-b-poly(L-lactic acid) Micelle after Oral Administration in Rats.

The purpose of this study was to evaluate the potential of micelle to change the pharmacokinetics of quercetin (QUT), with a primary goal of enhancing its oral bioavailability. QUT-loaded methoxy poly(ethylene glycol)-b-poly(L-lactic acid) micelle (QUT-loaded MPEG-b-PLLA micelle) was prepared by a thin-film hydration method, resulting in a particle size of 88.5 nm. A liquid chromatography tandem-mass spectrometry (LC-MS/MS) method was developed and validated for determination of QUT in rat plasma. The chromatographic separation was performed on an Agilent Eclipse-C18 (4.6 mm × 50 mm, 3.5 μm) with an isocratic mobile phase system consisting of water and methanol (30 : 70, v/v) at a flow rate of 0.4 mL/min. Calibration curves were linear over the concentration ranges of 2.5–2000 ng/mL for QUT. The micelle was orally administered at a single does in rats, and the pharmacokinetic parameters were evaluated and compared with that administered with the QUT aqueous suspension. The results show that the micelle was able to increase the QUT's oral bioavailability 9-fold compared to the QUT aqueous suspension. These results suggest that methoxy poly(ethylene glycol)-b-poly(L-lactic acid) is a potential carrier for the oral delivery of QUT.

Simultaneous determination of cucurbitacin IIa and cucurbitacin IIb of Hemsleya amabilis by HPLC-MS/MS and their pharmacokinetic study in normal and indomethacin-induced rats.

A selective and sensitive HPLC-MS/MS method was developed for the simultaneous determination of cucurbitacin IIa (cuIIa) and cucurbitacin IIb (cuIIb), the major bioactive cucurbitacins of Hemsleya amabilis, in rat plasma using euphadienol as internal standard (IS). After liquid-liquid extraction with dichloromethane, separation was achieved on a Syncronis HPLC C18 column (150 mm × 4.6 mm, 5 μm) using an isocratic mobile phase system consisting of acetonitrile-water (85:15, v/v) at a flow rate of 0.6 mL/min with a split ratio of 1:2. Detection was performed on a TSQ Quantum Ultra mass spectrometer equipped with an positive-ion electrospray ionization source. The lower limits of quantification (LLOQs) were 0.25 and 0.15 ng/mL for cuIIa and cuIIb, respectively. The intra- and inter-day precision was <11.5% for the LLOQs and each quality control level of the analytes, and accuracy was between -9.1 and 7.6%. The extraction recoveries of the analytes and IS from rat plasma were all >87.1%. The method was fully validated and applied to compare the pharmacokinetic profiles of the two cucurbitacins in rat plasma after oral administration of H. amabilis extract between normal and indomethacin-induced rats. Copyright © 2016 John Wiley & Sons, Ltd.

A rapid UPLC-MS/MS method for the determination of oleanolic acid in rat plasma and liver tissue: application to plasma and liver pharmacokinetics.

A reliable high-throughput ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for oleanolic acid (OA) determination in rat plasma and liver tissue using glycyrrhetic acid as the internal standard (IS). Plasma and liver homogenate samples were prepared using solid-phase extraction. Chromatographic separation was achieved on a C18 column using an isocratic mobile phase system. The detection was performed by multiple reaction monitoring mode via positive electrospray ionization interface. The calibration curves showed good linearity (R(2) > 0.9997) within the tested concentration ranges. The lower limit of quantification for plasma and liver tissue was ≤0.75 ng/mL. The intra- and inter-day precision and accuracy deviations were within ±15% in plasma and liver tissue. The mean extraction recoveries ranged from 80.8 to 87.0%. In addition, the carryover, matrix effect, stability and robustness involved in the method were also validated. The method was successfully applied to the plasma and hepatic pharmacokinetics of OA after oral administration to rats.

UHPLC/MS method for the estimation of ethambutol in human plasma and its application in tuberculosis patients.

Ultra Fast liquid chromatography – mass spectrometry method (UFLC-MS), for detection and quantification of the anti-tuberculosis drug, ethambutol (EMB), in human plasma is described here. The method uses a single Quadrapole mass spectrometer equipped with positive electrospray ionization detector, linked with Shimadzu 20AD ultrafast LC system. Ethambutol was extracted from human plasma (100 µl) through a single step, simple and straight forward acetonitrile deproteinization method, achieving 98 - 102 % analyte recovery. Short C18 column (50 mm x 2.1 mm) was used with isocratic mobile phase system consisting of 80% aqueous acetonitrile and 0.5% formic acid as additive. Selective ion monitoring (SIM) mode was selected with EMB m/z 205.3 in positive ion mode (MH+). This method achieved wide range of detection from microgram to nanograms of EMB (Linearity R value = 0.9999), with great precision and accuracy. The limits of quantification and detection were achieved upto 19.18 ng/ml and 5.78 ng/ml respectively. This method was applied successfully to determine EMB in plasma samples from TB patients who were receiving EMB along with other anti-TB drugs.

UPLC-MS/MS determination of voriconazole in human plasma and its application to a pharmacokinetic study.

A sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed to determine voriconazole in human plasma. Sample preparation was accomplished through a simple one-step protein precipitation with methanol. Chromatographic separation was carried out on an Acquity UPLC BEH C18 column using an isocratic mobile phase system composed of acetonitrile and water containing 1% formic acid (45:55, v/v) at a flow rate of 0.50 mL/min. Mass spectrometric analysis was performed using a QTrap5500 mass spectrometer coupled with an electrospray ionization source in the positive ion mode. The multiple reaction monitoring transitions of m/z 351.0 → 281.5 and m/z 237.1 → 194.2 were used to quantify voriconazole and carbamazepine (internal standard), respectively. The linearity of this method was found to be within the concentration range of 2.0-1000 ng/mL with a lower limit of quantification of 2.0 ng/mL. Only 1.0 min was needed for an analytical run. This fully validated method was successfully applied to the pharmacokinetic study after oral administration of 200 mg voriconazole to 20 Chinese healthy male volunteers.

UPLC-MS/MS Determination of Phentolamine in Human Plasma and its Application to a Pharmacokinetic Study

A sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed to determine phentolamine in human plasma. Sample preparation was accomplished through a simple liquid-liquid extraction with ethyl acetate. Chromatographic separation was carried out on an Acquity UPLC BEH C18 column using an isocratic mobile phase system composed of acetonitrile and 1% formic acid in water (33:67, v/v) at a flow rate of 0.45 mL/min. Mass spectrometric analysis was performed using a QTrap5500 mass spectrometer coupled with an electro-spray ionization (ESI) source in the positive ion mode. The MRM transitions of m/z 282.1 → 212.0 and m/z 237.1 → 194.2 were used to quantify for phentolamine and carbamazepine (internal standard, IS), respectively. The linearity of this method was found to be within the concentration range of 0.5-100.0 ng/mL with a lower limit of quantification of 0.5 ng/mL. Only 1.0 min was needed for an analytical run. This fully validated method was successfully applied to the pharmacokinetic study after oral administration of 60 mg phentolamine to 20 Chinese healthy male volunteers.

LC–ESI–MS method for the monitoring of Abl 1 tyrosine kinase

A liquid chromatography–electrospray ionization–mass spectrometric (LC–ESI–MS) method was developed and validated to study Abl 1 tyrosine kinase. An online desalting system was adopted, and a transformation of the ratio of product to substrate instead of a deuterated internal standard was introduced to calculate the concentration of product. In this study, the substrate used was Abltide (KKGEAIYAAPFA-NH2). The detection was performed by selected ion monitoring (SIM) mode via positive ESI interface. Chromatographic separation was achieved on a C18 column using an isocratic mobile phase system. The limit of quantification (LOQ) was 10nM for the product and 25nM for the substrate. The simple ratios of product to substrate maintained a linear relationship (R2=0.9997) over the ratio of 0–50% product. Intra- and inter-day precision was less than 10% and accuracy was from −1.6 to +5.3%. The validated method was applied to the Abl 1 kinase kinetic study and the Km and Vmax constants obtained for Abltide were 34.78μM and 5.563μmol/mg/min and for adenosine triphosphate (ATP) were 43.61μM and 5.906μmol/mg/min. The enzymatic reaction of Abl 1 tyrosine kinase belongs to ternary-complex mechanism.

Development of a HPLC/MS/MS method for simultaneous determination of tinidazole, dyclonine and chlorhexidine in rat plasma and its application in the pharmacokinetic research of a film-forming solution

A rapid and sensitive HPLC/MS/MS method was developed and validated for simultaneous determination of tinidazole, dyclonine and chlorhexidine, the three main components of a film-forming solution, in rat plasma. The plasma samples were pretreated by solid phase extraction (SPE) method. Separation was achieved on a Phenomenex Gemini C18 column (50mm×2.0mm, 5μm) using an isocratic mobile phase system composed of methanol-ammonium formate (10mM)-formic acid (56:44:0.2, v/v/v) (pH 3.5) at a flow rate of 0.2mL/min. Analytes were determined by tandem mass spectrometry with electrospray positive ionization and multiple-reaction monitoring (MRM) mode. The monitoring ions were (m/z) 247.4→(m/z) 81.9 for tinidazole, (m/z) 290.1→(m/z) 97.8 for dyclonine, (m/z) 505.0→(m/z) 335.3 for chlorhexidine and (m/z) 282.1→(m/z) 212.0 for phentolamine (internal standard). The calibration curves were linear in the range of 2–1000ng/mL for the three components. The precision and accuracy of the method were well within the generally accepted criteria for biomedical analysis. It has been successfully applied to the pharmacokinetic research of a film-forming solution in rat.