Liquid-liquid Extraction of Everolimus an Immunosuppressant from Human Whole Blood and its Sensitive Determination by UHPLC-MS/MS

A sensitive and precise ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method has been developed and fully validated for therapeutic drug monitoring of everolimus in human whole blood. Sample preparation involved liquid-liquid extraction of everolimus and its deuterated internal standard (IS, everolimus-d4) from 100 µL blood sample using diethyl ether: ethyl acetate (30:70, v/v) solvent system under alkaline conditions. The chromatography was conducted on a COSMOSIL 2.5C18-MS-II (50 mm × 2.0 mm, 2.5 µm) analytical column. The analyte and IS were eluted within 2.5 min under isocratic conditions using 10mM ammonium acetate, pH 6.00 adjusted with formic acid and acetonitrile (20:80, v/v). Multiple reaction monitoring was used for the quantitation of everolimus (m/z 975.5 → 908.5) and everolimus-d4 (m/z 979.6 → 912.6) in the positive ionization mode. The method was shown to be linear over the entire concentration range from 0.10-50.0 ng/mL. The recovery ranged from 90.9-94.8 % for everolimus and 91.4-95.6 % for everolimus-d4. The selectivity of the method is demonstrated in six different sources of blank human blood. The method is free from matrix effect as apparent from the post-column analyte infusion experiment, absolute and relative matrix effect results. The stability of everolimus in whole blood was thoroughly established under different storage conditions

New Improved UPLC-MS/MS Method for Reliable Determination of Clarithromycin in Human Plasma to Support a Bioequivalence Study

An improved, highly sensitive UPLC-MS/MS method has been developed for the determination of clarithromycin in human plasma. For sample preparation, liquid-liquid extraction with n-hexane: methyl tert-butyl ether (20:80, v/v) mixture was carried out using clarithromycin 13C-d3 as the internal standard (IS). Acquity UPLC BEH C18 (50 mm × 2.1 mm, 1.7 µm) analytical column was used for chromatography with methanol-5.0 mM ammonium formate, pH 3.0 (78:22, v/v) as the mobile phase under isocratic conditions. The analysis time was 1.5 min. Quantitation of analyte was done by tandem mass spectrometer using electrospray ionization in the positive mode. The precursor → product ion transitions monitored for clarithromycin and IS were m/z 748.9 → 158.1 and m/z 752.8 → 162.0 respectively. The method was validated over a dynamic concentration range of 0.80-1600 ng/mL with correlation coefficient (r2) ≥ 0.9998. The mean extraction recovery of clarithromycin was 96.2 % across six quality control levels. Intra-batch and inter-batch accuracy and precision (% CV) ranged from 96.8 to 103.5 % and 1.28 to 4.85 % respectively. Stability of clarithromycin in plasma was evaluated under different conditions like bench top, auto sampler, dry and wet extract, freeze-thaw and long term. The present method was successfully applied to a bioequivalence study in 20 healthy subjects who received single oral dose 250 mg clarithromycin tablet formulation. The reproducibility of the method was investigated by reanalysis of 100 incurred samples

Application of Lanthanide Polychelates Derived from 2,4-dihydroxy acetophenone Based Resin as Green Catalyst for Biginelli Reactions

In the present study, lanthanide polychelates of 2,4-dihydroxy acetophenone (DHAP) were evaluated for their applicability as green catalyst for various Biginelli reactions. Polymeric resins of DHAP (DHAP-1,2 PD, DHAP-1,3 PD, DHAP-1,3 BD and DHAP-1,4 BD) were synthesized and carried forward for the preparation of their polychelates with lanthanide metal ions viz. La3+, Pr3+, Nd3+, Sm3+, Gd3+, Tb3+ and Dy3+. The polychelates were assessed for their catalytic activity in the preparation of substituted 3,4-dihydropyrimidin-2(1H)-ones, largely known as the Biginelli compounds. In order to study the versatility and provide a comparative evaluation of the polychelates, a number of aldehyde derivatives and substituted 1,3-diketones were used. According to the results obtained, it was summarized that the polychelates possess desired catalytic effect for the aforementioned synthesis. The three component mixture of aldehyde, 1,3-dicarbonyl compound and urea were heated in presence of desired catalytic amount of lanthanide(III) polychelates to give corresponding 3,4-dihydropyrimidin-2(1H)-ones in 87-92 % yield. The reaction was completed within a reaction time of 2-6 h, and the polychelate catalysts could efficiently be reused for several times after simple washing step with distilled water

Sensitive and Rapid Determination of Gliclazide in Human Plasma by UPLC-MS/MS and its Application to a Bioequivalence Study

An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed for the determination of gliclazide in human plasma using gliclazide-d4 as the internal standard (IS). The plasma samples were prepared by protein precipitation with acetonitrile employing 50 µL human plasma. Chromatography was performed on Acquity UPLC BEH C18 (50 mm × 2.1 mm, 1.7 µm) analytical column under isocratic conditions using a mobile phase which consisted of 0.1% formic acid in water-acetonitAn ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed for the determination of gliclazide in human plasma using gliclazide-d4 as the internal standard (IS). The plasma samples were prepared by protein precipitation with acetonitrile employing 50 µL human plasma. Chromatography was performed on Acquity UPLC BEH C18 (50 mm × 2.1 mm, 1.7 µm) analytical column under isocratic conditions using a mobile phase which consisted of 0.1% formic acid in water-acetonitrile (10:90, v/v). The MRM transitions for gliclazide (m/z 324.2 → 127.3) and gliclazide-d4 (m/z 328.2 → 127.4) were monitored on a triple quadrupole mass spectrometer, operating in the positive ionization mode. The method was validated over a dynamic concentration range of 1.0-2000 ng/mL for gliclazide. Matrix effect was assessed by post-column analyte infusion and the mean extraction recovery was 95.7 % across six quality control levels. Stability of gliclazide in plasma was evaluated under different conditions like bench top, auto sampler, dry and wet extract, freeze-thaw and long term stability. The method was applied to a bioequivalence study with 30 mg gliclazide tablet formulation in 28 healthy subjects under fasting. Further, the assay reproducibility was confirmed by reanalysis of 129 incurred samples. rile (10:90, v/v). The MRM transitions for gliclazide (m/z 324.2 → 127.3) and gliclazide-d4 (m/z 328.2 → 127.4) were monitored on a triple quadrupole mass spectrometer, operating in the positive ionization mode. The method was validated over a dynamic concentration range of 1.0-2000 ng/mL for gliclazide. Matrix effect was assessed by post-column analyte infusion and the mean extraction recovery was 95.7 % across six quality control levels. Stability of gliclazide in plasma was evaluated under different conditions like bench top, auto sampler, dry and wet extract, freeze-thaw and long term stability. The method was applied to a bioequivalence study with 30 mg gliclazide tablet formulation in 28 healthy subjects under fasting. Further, the assay reproducibility was confirmed by reanalysis of 129 incurred samples

Validation of Simultaneous Quantitative Method of HIV Protease Inhibitors Atazanavir, Darunavir and Ritonavir in Human Plasma by UPLC-MS/MS

Objectives. HIV protease inhibitors are used in the treatment of patients suffering from AIDS and they act at the final stage of viral replication by interfering with the HIV protease enzyme. The paper describes a selective, sensitive, and robust method for simultaneous determination of three protease inhibitors atazanavir, darunavir and ritonavir in human plasma by ultra performance liquid chromatography-tandem mass spectrometry. Materials and Methods. The sample pretreatment consisted of solid phase extraction of analytes and their deuterated analogs as internal standards from 50 μL human plasma. Chromatographic separation of analytes was performed on Waters Acquity UPLC C18 (50 × 2.1 mm, 1.7 μm) column under gradient conditions using 10 mM ammonium formate, pH 4.0, and acetonitrile as the mobile phase. Results. The method was established over a concentration range of 5.0–6000 ng/mL for atazanavir, 5.0–5000 ng/mL for darunavir and 1.0–500 ng/mL for ritonavir. Accuracy, precision, matrix effect, recovery, and stability of the analytes were evaluated as per US FDA guidelines. Conclusions. The efficiency of sample preparation, short analysis time, and high selectivity permit simultaneous estimation of these inhibitors. The validated method can be useful in determining plasma concentration of these protease inhibitors for therapeutic drug monitoring and in high throughput clinical studies

Determination of mycophenolic acid in human plasma by ultra performance liquid chromatography tandem mass spectrometry

AbstractA simple, sensitive and high throughput ultra performance liquid chromatography tandem mass spectrometry method has been developed for the determination of mycophenolic acid in human plasma. The method involved simple protein precipitation of MPA along with its deuterated analog as an internal standard (IS) from 50µL of human plasma. The chromatographic analysis was done on Acquity UPLC C18 (100mm×2.1mm, 1.7µm) column under isocratic conditions using acetonitrile and 10mM ammonium formate, pH 3.00 (75:25, v/v) as the mobile phase. A triple quadrupole mass spectrometer operating in the positive ionization mode was used for quantitation. In-source conversion of mycophenolic glucuronide metabolite to the parent drug was selectively controlled by suitable optimization of cone voltage, cone gas flow and desolvation temperature. The method was validated over a wide concentration range of 15–15000ng/mL. The mean extraction recovery for the analyte and IS was >95%. Matrix effect expressed as matrix factors ranged from 0.97 to 1.02. The method was successfully applied to support a bioequivalence study of 500mg mycophenolate mofetil tablet in 72 healthy subjects

ESTIMATION OF CELECOXIB IN HUMAN PLASMA BY RAPID AND SELECTIVE LC-MS/MS METHOD FOR A BIOEQUIVALENCE STUDY

Objective: A selective, sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay has been developed for the determination of celecoxib (CXB) in negative ionization mode.Methods: Celecoxib and celecoxib-D7 (CXB-D7) as internal standard (IS) were extracted from 300 µl human plasma by solid-phase extraction using strata-X SPE cartridges. Chromatographic separation was achieved on ACE C8-300 (50 × 4.0 mm, 3.0 μm) column using methanol-1.0 mmol ammonium acetate solution in 80:20 (v/v) ratio. The protonated precursor to product ion transitions studied for CXB and CXB-D7 were m/z 380.0 → 315.9 and 387.0 → 323.0, respectively.Results: The limit of detection (LOD) and lower limit of quantitation of the method were 2.50 and 10.0 ng/ml respectively with a linear dynamic range of 10.0-4000 ng/ml for CXB. The intra-batch and inter-batch precision (% CV) and mean relative recovery across quality control levels is<7.2 % and 85.5 % respectively. Matrix effect in human plasma, expressed as IS-normalized matrix factor ranged from 0.99-1.03.Conclusion: The method was successfully applied in healthy subjects using a single dose of 400 mg celecoxib capsules under fasting and fed conditions. The reproducibility in the measurement of study data is demonstrated by incurred sample reanalysis

COMPARATIVE EVALUATION OF FIRST ORDER, ABSORBANCE RATIO AND BIVARIATE SPECTROPHOTOMETRIC METHODS FOR DETERMINATION OF ATOVAQUONE AND PROGUANIL IN PHARMACEUTICAL FORMULATION MALARONE®

Objective: Three simple, rapid, accurate and precise spectrophotometric methods have been developed for the simultaneous estimation of atovaquone and proguanil hydrochloride in pharmaceutical preparations.Methods: The determination of drugs was carried out using the first order derivative, absorbance-ratio and bivariate spectrophotometric methods. The methods were validated for their linearity, accuracy and precision, recovery and ruggedness according to the ICH guidelines.Results: The linearity was established in the concentration range of 1.0-10 µg/ml for atovaquone and 0.5-8.0 µg/ml for proguanil hydrochloride by all three methods. The limit of detection (LOD) and the limit of quantitation (LOQ) of the methods varied from 0.252 to 0.270 µg/ml and 0.764 to 0.825 µg/ml for atovaquone and 0.119 to 0.156 µg/ml and 0.361 to 472 µg/ml for proguanil hydrochloride respectively. The intra-and inter-batch accuracy (% recovery) and precision (% RSD) ranged from 99.16 to 101.05 % and 0.603 to 1.048 for atovaquone and 99.74 to 101.12 % and 0.593 to 1.001 for proguanil respectively.Conclusion: The proposed methods were applied to a pharmaceutical formulation with acceptable accuracy and precision without any interference from commonly used excipients and additives. The results show that all three methods are comparable, cost effective and rapid and thus can be readily used in quality control labs for routine analysis of these drugs.Â

DETERMINATION OF CAPECITABINE-AN ANTICANCER DRUG IN DRIED BLOOD SPOT BY LC-ESI-MS/MS

Objective: Capecitabine (Cape), the first oral prodrug which belongs to the group of fluoro pyrimidines is the most frequently prescribed anticancer drug for the treatment of metastatic breast and colorectal cancers. The article describes a selective and robust method for determination of Cape in dried blood spots (DBS) by liquid chromatography-tandem mass spectrometry (LC-MS/MS).Methods: Cape fortified DBS was punched and extracted with ethyl acetate using capecitabine-d11 as the internal standard (IS). Chromatographic separation of Cape and IS from endogenous matrix was performed on Phenomenex Gemini C18 (150 × 4.6 mm, 5mm) column under isocratic condition using acetonitrile: 2 mmol ammonium formate (pH 3.0, adjusted with 0.1 % formic acid) (80:20, v/v) as the mobile phase. Detection and quantification were carried on a triple quadrupole mass spectrometer, using electro spray ionization technique in the positive ionization mode.Results: The method was established over a concentration range of 10-10000 ng/ml. Accuracy, precision, selectivity, recovery, matrix effect and stability of the analyte were also estimated and the results were within the acceptance criteria. Further, precise results were obtained using an optimum spot volume of 10 µl with good spot homogeneity. Blood samples with hematocrit values varying from 24 % to 45 % gave acceptable results with good accuracy and precision.Conclusion: The efficiency of dried blood spot sample preparation, short analysis time and high selectivity permits estimation of Cape in a small blood volume. The validation results suggest that the method is precise, accurate, and reproducible and can be useful in therapeutic drug monitoring of Cape.Â

SENSITIVE AND RAPID ESTIMATION OF LAPATINIB, AN ANTICANCER DRUG IN SPIKED HUMAN PLASMA BY LC-MS/MS

Objective: The work presents a sensitive, selective and rapid determination of lapatinib, a potent anticancer drug in human plasma by liquid chromatography-tandem mass spectrometry.Methods: Liquid-liquid extraction of lapatinib and lapatinib-d4, added as an internal standard (IS) was carried out from 100 µl plasma sample. Chromatographic analysis was performed on ACE C18 (100 mm × 4.6 mm, 5 µm) column using 10 mmol ammonium formate buffer (pH 3.5) and acetonitrile (10:90, v/v) as the mobile phase. The precursor ion → product ion transitions for lapatinib (m/z 581.1 → 365.2) and IS (m/z 585.1 → 365.0) were monitored on a triple quadrupole mass spectrometer in the positive electrospray ionization mode. The method was validated in accordance with the US FDA guidelines.Results: A linear concentration range was established from 2.50-2500 ng/ml for lapatinib. The intra-batch and inter-batch precision were ≤ 4.81 %. The recovery of lapatinib and IS from plasma samples ranged from 88.7 to 95.8 % and 85.9 to 96.5 % respectively. The accuracy and precision (% CV) for the stability of lapatinib under different storage conditions showed a variation from 95.2 to 102.2 % and 1.19 to 4.35 % respectively at low and high QC levels. Under optimized chromatographic conditions, the retention time for lapatinib was 1.406 min with a total run time of 2.5 min for each sample.Conclusion: The validation results demonstrate that the method is simple, accurate, precise and reproducible. The developed method can be readily used for pharmacokinetics/bioequivalence studies in patients as well as healthy subjects.Â