ABSTRACT
Objective: To develop and validate a sensitive liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS/MS) technique for the quantification of dapagliflozin and saxagliptin in plasma by linagliptin as internal standard. Methods: Chromatography was achieved on hypersil C18 (50 mmx4 mm) 5 µ analytical column with 0.1% formic acid and acetonitrile (25:75 V/V) as mobile phase at 0.7 ml/min flow rate. Dapagliflozin, saxagliptin, and linagliptin were detected at m/z 409.14/135.0, m/z 316.2/180.13 and m/z 472.54/456.21 respectively. Drugs and internal standard were extracted by LLE (liquid-liquid extraction). Results: Developed technique was validated over 0.5-1500.0 ng/ml linear concentration range for dapagliflozin and 2.00-2000.0 ng/ml for saxagliptin. This method established with intra-batch and inter-batch precision within 2.44-8.12% and 1.25-7.14 % for dapagliflozin and 1.84-7.5 % and 1.02–6.00 % for saxagliptin. This method established with intra-batch and inter-batch accuracy for dapagliflozin within 98.86-103% and 96.98-102 % respectively and for saxagliptin within 98.05-109.06 % and 97.00-104.00 % respectively. Conclusion: Both dapagliflozin and saxagliptin were stable during three freeze-thaw cycles, long term and bench-top stability studies. The developed method was useful for the routine analysis of dapagliflozin and saxagliptin simultaneously in plasma samples.
ABSTRACT
In order to study the pesticide residues of the medicinal Crataegi Fructus,this study aims to establish an analysis method for pesticide residues( mainly containing insecticides and fungicides) suitable for the actual situation of medicinal Crataegi Fructus based on the survey of the pesticides of the Crataegi Fructus base,combined with the blind screening results of the LC-ESI-MS/MS pesticide screening platform established by the research team in the early stage. Then,the pesticide residues in medicinal Crataegi Fructus from Shandong,Hebei,Henan,Shanxi,and Liaoning( main cultivation areas) were analyzed. The samples were pretreated by the modified Qu ECh ERS method,i.e.,extracted with acetonitrile-water( 9 ∶1),purified by PSA,C_(18),GCB,silica gel. The detection of pesticides was performed by LC-MS/MS. The ion source was ESI with positive scanning mode,and the linearity of 11 kinds of pesticides in the range of 5-300 μg·kg~(-1) was acceptable( R~2>0. 996 9). All the recoveries of pesticides were within 70. 02%~(-1)12. 0% in the low,medium and high levels,with RSD≤17%. The results showed that the detection rate of carbendazim,chlorpyrifos and difenoconazole is 79%,82%,56%,respectively. Besides,the prohibition pesticide carbofuran were detected in some of the batches,indicating the security risk. This study provides methodological references and basic data for risk assessment of Crataegi Fructus and government regulation.
Subject(s)
Chromatography, Liquid , Crataegus/chemistry , Drug Contamination , Drugs, Chinese Herbal/analysis , Pesticide Residues/analysis , Surveys and Questionnaires , Tandem Mass SpectrometryABSTRACT
A highly sensitive, rapid and rugged liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) method was developed for reliable estimation of amantadine (AMD), an antiviral drug in human plasma. The analyte and internal standard (IS), amantadine-d6 (AMD-d6), were extracted from 200 μL plasma by solid phase extraction on Phenomenex Strata-X-C 33 μ cartridges. Chromatography was performed on Synergi? Hydro-RP C18 (150 mm × 4.6 mm, 4 μm) analytical column using a mixture of acetonitrile and 10 mM ammonium formate, pH 3.0 (80:20, v/v) as the mobile phase. Detection and quantitation was done by multiple reaction monitoring in the positive ionization mode for AMD (m/z 152.1 → 135.1) and IS (m/z 158.0 → 141.1) on a triple quadrupole mass spectrometer. The assay was linear in the concentration range of 0.50–500 ng/mL with correlation coefficient (r2) ≥ 0.9969. The limit of detection of the method was 0.18 ng/mL. The intra-batch and inter-batch precisions were ≤ 5.42% and the accuracy varied from 98.47% to 105.72%. The extraction recovery of amantadine was precise and quantitative in the range of 97.89%–100.28%. IS-normalized matrix factors for amantadine varied from 0.981 to 1.012. The stability of AMD in whole blood and plasma was evaluated under different conditions. The developed method was successfully applied for a bioequivalence study with 100 mg of AMD in 32 healthy volunteers. The re-producibility of the assay was determined by reanalysis of 134 subject samples.
ABSTRACT
Zidvovudine (AZT) is a nucleoside analogue reverse transcriptase inhibitor (NRTI), a class of anti-retroviral drug. A stability-indicating assay method for AZT was developed in line with ICH guideline. Successful separation of AZT and its degradation products was achieved by gradient elution mode on reverse phase C18 column using 10 mM ammonium acetate: acetonitrile as the mobile phase at 0.8 mL/min flow rate, 25 μL injection volume, 30 °C column temperature and 285 nm detection wavelength. Two major acid degradation products were identified and characterized by liquid chromatography–electrospray ionization mass spectro-metry (LC–ESI/MS/MS) and accurate mass measurements. The probable mechanisms for the formation of degradation products were identified based on a comparison of the fragmentation pattern of the [M + H] + ions of AZT and its degradation products. One of the degradation products, DP-1, was isolated by semi-preparative high performance liquid chromatography (HPLC) using Waters XBridge Prep C18 (250 mm×10 mm, 5 μm). Degradation products showed higher toxicity compared to the drug in some models assessed by TOPKAT software. The method validation was performed with respect to robustness, specificity, linearity, precision and accuracy as per ICH guideline Q2 (R1).
ABSTRACT
Antecedentes. El aldicarb es un plaguicida carbamato de alta toxicidad asociado a intoxicaciones agudas fatales en el ser humano. Su mecanismo de acción consiste en la inhibición de la enzima acetilcolinesterasa (AChE) que ocasiona la acumulación del neurotransmisor acetilcolina en la hendidura sináptica. Esta acumulación provoca síntomas colinérgicos y, dependiendo de la dosis de exposición, puede paralizar los sistemas respiratorio y nervioso hasta llegar a la muerte. Objetivo. Determinar el nivel de aldicarb en sangre post mortem en casos de intoxicación aguda. Materiales y métodos. Investigación de tipo experimental empleando un cromatógrafo líquido con espectrometría de masas, con ionización electrospray y análisis en modo tándem (LC-ESI-MS/MS). Los estándares de aldicarb y el aldicarb-d3 fueron comprados de Dr. Ehrenstorfer GmbH. El método consiste en una precipitación de proteínas de la sangre y su posterior análisis por LC-ESI-MS/MS, utilizando el aldicarb-d3 como estándar interno. El método fue aplicado a siete casos de intoxicación letal por presunta acción del aldicarb. Resultados. El aldicarb se encontró en la sangre de seis de los casos estudiados, en niveles desde 0.12 a 1.90 µg/mL. Solo en uno de los casos no se detectó aldicarb. En cuanto la presunta manera de muerte, en seis de los casos analizados fue el suicidio y en un caso se clasificó como muerte en estudio. Conclusiones. Los resultados obtenidos con la metodología analítica y la técnica LC-ESI-MS/MS son satisfactorios en términos de la determinación cuantitativa de aldicarb en sangre total post mortem. La aplicación de la metodología descrita en toxicología forense evidencia el empleo de este plaguicida en actos suicidas.
Background. Aldicarb is a high toxicity carbamate pesticide associated to human fatal acute intoxications. Its mechanism of action consists of the inhibition of the acetylcholinesterase enzyme (AChE), which induces the accumulation of the neurotransmitter acetylcholine in the synaptic cleft. This accumulation induces cholinergic symptoms and, depending on the exposition dose, it can paralyze the respiratory and nervous systems, leading to death. Objective. To determine aldicarb levels in post mortem blood in cases of acute intoxication. Materials and methods. An experimental research was conducted using liquid chromatography tandem-mass spectrometry (LC-ESI-MS/MS) with electrospray ionization. The aldicarb and aldicarb-d3 standards were purchased from Dr. Ehrenstorfer GmbH corporation. This method carries out a protein precipitation of blood and its analysis using LC-ESI-MS/MS, using aldicarb-d3 as internal standard. This method was applied to seven cases of fatal intoxication by presumable action of aldicarb. Results. Aldicarb was found in six of the studied cases on levels between 0.12 and 1.90 µg/mL. Aldicarb was not detected in blood only in one case. Six of the cases were associated to suicide as a manner of death and in one of them it remained under study. Conclusions. The results obtained with the analytical methodology and the use of the LC-ESI-MS/MS technique are satisfactory in terms of the quantitative determination of aldicarb in post mortem total blood. The application of the described methodology in forensic toxicology evidences the use of this pesticide in suicidal practices.
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A sensitive, selective and high-throughput liquid chromatography–tandem mass spectrometry (LC–ESI–MS/MS) method was developed and validated for the quantitation of tolvaptan in rabbit plasma. Sample clean-up involved liquid–liquid extraction (LLE) and chromatography was performed on Zorbax SB C18 analytical column (50 mm ? 2.1 mm, 3.5 mm) using 0.1%formic acid:methanol (20:80, v/v) as the mobile phase. The parent-product ion transitions for the drug (m/z 449.2-252.1) and IS (m/z 456.2-259.2) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) and positive ion mode. The method was validated over the concentration range of 0.10–1000.00 ng/mL and successfully applied to a pharmacokinetic study of healthy rabbits.
ABSTRACT
A selective and rapid high-performance liquid chromatography–tandem mass spectrometry method was developed and validated for the quantification of raltegravir using raltegravir-d3 as an internal standard (IS). The analyte and IS were extracted with methylene chloride and n-hexane solvent mixture from 100 mL human plasma. The chromatographic separation was achieved on a Chromolith RP-18e endcapped C18 (100 mm ? 4.6 mm) column in a run time of 2.0 min. Quantitation was performed in the negative ionization mode using the transitions of m/z 443.1-316.1 for raltegravir and m/z 446.1-319.0 for IS. The linearity of the method was established in the concentration range of 2.0–6000 ng/mL. The mean extraction recovery for raltegravir and IS was 92.6% and 91.8%, respectively, and the IS-normalized matrix factors for raltegravir ranged from 0.992 to 0.999. The application of this method was demonstrated by a bioequivalence study on 18 healthy subjects.
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Objective: To develop a sensitive and rapid liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the simultaneous determination of wogonin, coptisine, berberine, palmatine, jatrorrhizine, phellodendrine, magnoflorine, and wogonoside in rat plasma and to evaluate the pharmacokinetic characteristics of Huanglian Jiedu Decoction (HJD). Methods: LC separation was performed on an Acquity HSS T3 column (100 mm × 2.1 mm, 1.8 μm) using gradient elution with the mobile phase consisting of acetonitrile and 0.1% formic acid water. The detection was accomplished by using positive electrospray ionization in multiple-reaction monitoring mode. Plasma samples were pretreated by protein precipitation. Results: The method showed a good linearity over a wide concentration range (r2 > 0.99). The lower limits of quantification were 0.20 ng/mL for coptisine and phellodendrine, 0.48 ng/mL for berberine, 0.10 ng/mL for jatrorrhizine, 0.32 ng/mL for magnoflorine, 0.30 ng/mL for palmatine, and 4.80 ng/mL for wogonin and wogonoside, respectively. The intra- and inter-day precision of the analytes was less than 12.11%, while the accuracy was between -14.46% and 4.86%. The mean recovery of all the analytes ranged from 93.10% to 110.91%. Conclusion: This validated method offers the advantages of high sensitivity. It is successfully applied to evaluating the pharmacokinetic properties of HJD. © 2014 Tianjin Press of Chinese Herbal Medicines.
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Verapamil is a synthetic derivative of papaverine, which is used therapeutically as a hypertensive, antiarrhythmic and antianginal. This study describes an analytical method for the determination of verapamil in biological matrices of blood and urine, which consists of a liquid-liquid extraction of samples for analysis using liquid chromatography-mass spectrometry (LC-ESI-MS/ MS), with flurazepam as an internal standard. The method was applied to the acute fatal intoxication of a 17-year-old young woman who consumed 170 tablets of verapamil; the concentration of this medication found in the blood was 18.261mg/L and 0.369mg/L in the urine. This study also puts forth the use of LC-ESI-MS/MS in the analysis of verapamil in biological samples for applications in forensic toxicology.
El verapamilo es un derivado sintético de la papaverina que se emplea a nivel terapéutico como antihipertensivo, antiarrítmico y antianginoso. El presente estudio describe un método analítico para la determinación de verapamilo en las matrices biológicas sangre y orina, que consiste en una extracción líquido-líquido de las muestras, para luego ser analizadas por cromatografía líquida con ionización electro-spray y espectrometría de masas tándem (LC-ESI-MS/MS), empleando el flurazepam como estándar interno. El método desarrollado fue aplicado a la intoxicación aguda fatal de una joven de 17 años de edad quien consumió 170 tabletas de verapamilo; la concentración de este medicamento encontrada en sangre fue de 18.261mg/L y de 0.369mg/L en la orina. De igual forma, este estudio expone la utilidad de emplear LC-ESI-MS/ MS en el análisis de verapamilo en muestras biológicas para su aplicación en toxicología forense.
Subject(s)
Humans , Forensic Toxicology , Verapamil , Chromatography, LiquidABSTRACT
The peptides produced enzymatically from various plants have shown various biological activities including cytotoxicity. Different types of cytotoxic peptides have been reported from the seeds and leaves of Violaceae, Rubiaceae and Annonaceae families. In this study, we report purification and characterization of peptide(s) showing cytotoxic activity against A549 and HeLa cancer cell lines from the seeds of Polyalthia longifolia (Annonaceae). Seed proteins of P. longifolia were extracted and hydrolyzed using trypsin. The enzyme hydrolysate was applied on to a Sephadex G10 column and eluted using Tris-HCl buffer (pH 7.5). Two fractions F1 and F2 were obtained, of which F2 showed significant cytotoxic activity against lung (A549) cancer cells at 10 µg/mL and cervical (HeLa) cancer cell lines at 30 µg/mL, as revealed by the MTT assay. DNA fragmentation was observed in the tested cancer cell lines treated with F2 peptide at a concentration of 10 µg/mL and 30 µg/mL, respectively. Further, increased number of apoptotic cells was observed in sub-G0 phase of cell cycle of A549 and HeLa cell lines, when treated with 10 µg/mL and 30 µg/mL of F2, as revealed by the flow cytometric analyses. FTIR spectrum of F2 peptide detected the presence of stretching vibrations of carboxylic acid OH residue with peak at 3420 cm-1 and carbonyl (C=O) groups at 1636 cm-1, respectively. RP-HPLC analysis of F2 peptide showed a single peak at a retention time of 12.8 min detected at 280 nm, depicting the purity of F2 to be more than 90%. LC-ESI-MS/MS analysis showed the average theoretical mass of F2 to be 679.8 using m/z ratios. In conclusion, the findings suggest that F2 peptide is an effective inducer of apoptosis of cancer cells, thus offers an important strategy in the development of cancer therapeutics.
Subject(s)
Apoptosis/drug effects , Cell Cycle/drug effects , Cell Proliferation/drug effects , Chromatography, High Pressure Liquid , HeLa Cells , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Peptide Fragments/isolation & purification , Peptide Fragments/pharmacology , Polyalthia/chemistry , Seeds/chemistry , Spectrometry, Mass, Electrospray Ionization , Spectroscopy, Fourier Transform Infrared , Tandem Mass Spectrometry , Tumor Cells, CulturedABSTRACT
A selective, sensitive and high throughput liquid chromatography-tandem mass spectro-metry (LC-ESI-MS/MS) method has been developed for separation and quantification of metoprolol enantiomers on a chiral Lux Amylose-2 (250 mm×4.6 mm, 5 mm) column. Solid phase extraction of (S)-(-)- and (R)-(t)-metoprolol and rac-metoprolol-d6 as an internal standard (IS) was achieved on Lichrosep DVB HL cartridges employing 200 mL human plasma. Both the analytes were chromatographically separated with a resolution factor of 2.24 using 15 mM ammonium acetate in water, pH 5.0 and 0.1% (v/v) diethyl amine in acetonitrile (50:50, v/v) as the mobile phase within 7.0 min. The precursor-product ion transitions for the enantiomers and IS were monitored in the multiple reaction monitoring and positive ionization mode. The method was validated over the concentration range of 0.500-500 ng/mL for both the enantiomers. Matrix effect was assessed by post-column analyte infusion experiment and the mean extraction recovery was greater than 94.0% for both the enantiomers at all quality control levels. The stability of analytes was evaluated in plasma and whole blood under different storage conditions. The method was successfully applied to a clinical study in 14 healthy volunteers after oral administration of 200 mg metoprolol tablet under fasting conditions. The assay reproducibility is shown by reanalysis of 68 incurred samples. The suitability of the developed method was assessed in comparison with different chromatographic methods developed for stereoselective analysis of metoprolol in biological matrices.
ABSTRACT
Obesity can be considered as a chronic illness of epidemic proportion and its incidents have increased exponentially in recent years.The use of anti-obesity drugs such as sibutramine is somewhat helpful.There is a need to quantify such drugs in biological samples,which is generally quite difficult.In this report,we developed and validated a simple,sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of sibutramine (SB) and its two metabolites N-des methyl sibutramine (DSB) and N-di desmethyl sibutramine (DDSB) in human plasma.Zorbax SBC18 (4.6 mm × 75 mm,3.5 μm,80 (A)) analytical column and 5 mM ammonium formate:acetonitrile (10∶90,v/v) mobile phase were used for chromatographic separation of SB,DSB and DDSB.Multiple reaction monitoring (MRM) in the positive mode was used to detect SB,DSB and DDSB at m/z 280.3/124.9,266.3/125.3 and 252.2/124.9,respectively.Liquid liquid extraction was used for the extraction of analytes and internal standard from human plasma.This method was validated over a linear concentration range of 10.0-10,000.0 pg/mL for SB,DSB and DDSB with correlation coefficients (r) of ≥0.9997.The drug and the two metabolites were stable in plasma samples.The validated method was successfully applied in a bioequivalence and pharmacokinetic study with human volunteers under fasting condition.
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A sensitive and reliable liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS) method was established to simultaneously quantitate four categories of compounds (isoflavonoids, flavonoids, alkaloids and saponins) in Gegen-Qinlian decoction (GQD). These compounds were separated by a Shiseido CAPCELL PAK C18 column with a linear gradient consisting of 0.1% (v/v) formic acid in water (A) and 0.1% (v/v) formic acid in acetonitrile (B), and delivered at a flow rate of 0.3 mL/min. All the analytes were determined by electrospray positive ionization tandem mass spectrometry in a multiple reaction monitoring (MRM) mode. Linearity, accuracy, precision, recovery and stability of the method were evaluated with the validation over the range of 4.0-538 5 ng/mL. The proposed method was applied to the analysis of a Chinese herbal preparation GQD successfully.
ABSTRACT
A sensitive and reliable liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/ MS) method was established to simultaneously quantitate four categories of compounds (isoflavonoids, flavonoids, alkaloids and saponins) in Gegen-Qinlian decoction (GQD). These compounds were separated by a Shiseido CAPCELL PAK C18 column with a linear gradient consisting of 0.1% (v/v) formic acid in water (A) and 0.1% (v/v) formic acid in acetonitrile (B), and delivered at a flow rate of 0.3 mL/min. All the analytes were determined by electrospray positive ionization tandem mass spectrometry in a multiple reaction monitoring (MRM) mode. Linearity, accuracy, precision, recovery and stability of the method were evaluated with the validation over the range of 4.0-5385 ng/mL. The proposed method was applied to the analysis of a Chinese herbal preparation CQD successfully.