Simultaneous determination of telmisartan and amlodipine in human plasma by LC–MS/MS and its application in a human pharmacokinetic study

A rapid and sensitive liquid chromatography–tandem mass spectrometric (LC–MS/MS) assay method has been developed and fully validated for the simultaneous quantification of telmisartan and amlodipine in human plasma. Carbamazepine was used as an internal standard. Analytes and the internal standard were extracted from human plasma by solid-phase extraction technique using Waters Oasis^® HLB 1 cm³ (30 mg) extraction cartridge. The reconstituted samples were chromatographed on a Hypurity advance C₁₈ column (50 mm×4.6 mm, 5 μm) using a mixture of acetonitrile–5 mM ammonium acetate buffer (pH-4.0) (50:50, v/v) as the mobile phase at a flow rate of 0.8 mL/min. The calibration curve obtained was linear (r≥0.99) over the concentration range of 2.01–400.06 ng/mL for telmisartan and 0.05–10.01 ng/mL for amlodipine. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. A run time of 2.5 min for each sample made it possible to analyze more than 400 human plasma samples per day. The proposed method was found to be applicable to clinical studies.     

Simultaneous determination of borneol and its metabolite in rat plasma by GC–MS and its application to pharmacokinetic study

A gas chromatography mass spectrometry (GC–MS) method has been developed and fully validated for the simultaneous determination of natural borneol (NB) and its metabolite, camphor, in rat plasma. Following a single liquid–liquid extraction, the analytes were separated using an HP-5MS capillary column (0.25 mm×30 m×0.25 μm) and analyzed by MS in the selected ion monitoring mode. Selected ion monitor (m/z) of borneol, camphor and internal standard was 95, 95 and 128, respectively. Linearity, accuracy, precision and extraction recovery of the analytes were all satisfactory. The method was successfully applied to pharmacokinetic studies of NB after oral administration to Wistar rats.     

Quantification of sibutramine and its two metabolites in human plasma by LC–ESI-MS/MS and its application in a bioequivalence study

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 SB-C18 (4.6 mm×75 mm, 3.5 μm, 80 Å) 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.     

Simultaneous determination of ezetimibe and simvastatin in rat plasma by stable-isotope dilution LC-ESI–MS/MS and its application to a pharmacokinetic study

A simple, sensitive and specific liquid chromatography–tandem mass spectrometry method was developed for simultaneous quantification of ezetimibe and simvastatin in rat plasma. The deuterium isotopes: ezetimibe d₄ and simvastatin d₆ were used as internal standards for ezetimibe and simvastatin, respectively. MS/MS detection involved a switch of electron spray ionization mode from negative to positive at retention time 3.01 min. Samples were extracted from plasma by liquid–liquid extraction using tertiary butyl methyl ether. Chromatographic separation was achieved with Agilent Eclipse XBD-C₁₈ column using mobile phase that consisted of a mixture of ammonium acetate (pH4.5; 10 mM)–acetonitrile (25:75 v/v). The method was linear and validated over the concentration range of 0.2–40.0 ng/mL for simvastatin and 0.05–15.0 ng/mL for ezetimibe. The transitions selected were m/z 408.3→271.1 and m/z 412.0→275.10 for ezetimibe and ezetimibe d₄, and m/z 419.30→285.20 and m/z 425.40→199.20 for simvastatin and simvastatin d₆. Intra- and inter-batch precisions for ezetimibe were 1.6–14.8% and 2.1–13.4%; and for simvastatin 0.94–9.56% and 0.79–12%, respectively. The proposed method was sensitive, selective, precise and accurate for the quantification of ezetimibe and simvastatin simultaneously in rat plasma. The method was successfully applied to a pharmacokinetic study by oral co-administration of ezetimibe and simvastatin in SD rats.     

Validated LC–MS/MS method for simultaneous determination of SIM and its acid form in human plasma and cell lysate: Pharmacokinetic application

Simvastatin (SIM) is a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor widely used in hyperlipidemia therapy. SIM has recently been studied for its anticancer activity at doses higher than those used for the hyperlipidemia therapy. This prompted us to study the pharmacokinetics of high-dose SIM in cancer patients. For this purpose, an LC–MS/MS method was developed to measure SIM and its acid form (SIMA) in plasma and peripheral blood mononuclear cells (PBMCs) obtained from patients. Chromatographic analyte separation was carried out on a reverse-phase column using 75:25 (% v/v) acetonitrile:ammonium acetate (0.1 M, pH 5.0) mobile phase. Detection was performed on a triple quadrupole mass spectrometer, equipped with a turbo ion spray source and operated in positive ionization mode. The assay was linear over a range 2.5–500 ng/mL for SIM and 5–500 ng/mL for SIMA in plasma and 2.5–250 ng/mL for SIM and 5–250 ng/mL for SIMA in cell lysate. Recovery was >58% for SIM and >75% for SIMA in both plasma and cell lysate. SIM and SIMA were stable in plasma, cell lysate and the reconstitution solution. This method was successfully applied for the determination of SIM and SIMA in plasma and PBMCs samples collected in the pharmacokinetic study of high-dose SIM in cancer patients.     

Improved simultaneous quantitation of candesartan and hydrochlorthiazide in human plasma by UPLC–MS/MS and its application in bioequivalence studies

A validated ultra-performance liquid chromatography mass spectrometric method (UPLC–MS/MS) was used for the simultaneous quantitation of candesartan (CN) and hydrochlorothiazide (HCT) in human plasma. The analysis was performed on UPLC–MS/MS system using turbo ion spray interface. Negative ions were measured in multiple reaction monitoring (MRM) mode. The analytes were extracted using a liquid–liquid extraction (LLE) method by using 0.1 mL of plasma volume. The lower limit of quantitation for CN and HCT was 1.00 ng/mL whereas the upper limit of quantitation was 499.15 ng/mL and 601.61 ng/mL for CN and HCT respectively. CN d₄ and HCT-¹³Cd₂ were used as the internal standards for CN and HCT respectively. The chromatography was achieved within 2.0 min run time using a C18 Phenomenex, Gemini NX (100 mm×4.6 mm, 5 µm) column with organic mixture:buffer solution (80:20, v/v) at a flow rate of 0.800 mL/min. The method has been successfully applied to establish the bioequivalence of candesartan cilexetil (CNC) and HCT immediate release tablets with reference product in human subjects.     

Precolumn derivatization LC–MS/MS method for the determination and pharmacokinetic study of glucosamine in human plasma and urine

A selective precolumn derivatization liquid chromatography–tandem mass spectrometric (LC–MS/MS) method for the determination of glucosamine in human plasma and urine has been developed and validated. Glucosamine was derivatized by o-phthalaldehyde/3-mercaptopropionic acid. Chromatographic separation was performed on a Phenomenex ODS column (150 mm×4.6 mm, 5 μm) using linear gradient elution by a mobile phase consisting of methanol (A), and an aqueous solution containing 0.2% ammonium acetate and 0.1% formic acid (B) at a flow rate of 1 mL/min. Tolterodine tartrate was used as the internal standard (IS). With protein precipitation by acetonitrile and then the simple one-step derivatization, a sensitive bio-assay was achieved with the lower limit of quantitation (LLOQ) as low as 12 ng/mL for plasma. The standard addition calibration curves suitable for clinical sample analysis showed good linearity over the range of 0.012–8.27 μg/mL in plasma and 1.80–84.1 μg/mL in urine. The fully validated method has been successfully applied to a pharmacokinetic study of compound glucosamine sulfate dispersible tablets in health Chinese volunteers receiving single oral doses at 500, 1000 and 1500 mg of glucosamine sulfate, as well as multiple oral doses of 500 mg t.i.d. for 7 consecutive days.     

Selective separation,detection of zotepine and mass spectral characterization of degradants by LC–MS/MS/QTOF

A simple, precise, accurate stability-indicating gradient reversed-phase high-performance liquid chromatographic (RP–HPLC) method was developed for the quantitative determination of zotepine (ZTP) in bulk and pharmaceutical dosage forms in the presence of its degradation products (DPs). The method was developed using Phenomenex C₁₈ column (250 mm×4.6 mm i.d., 5 µm) with a mobile phase containing a gradient mixture of solvents, A (0.05% trifluoroacetic acid (TFA), pH=3.0) and B (acetonitrile). The eluted compounds were monitored at 254 nm; the run time was within 20.0 min, in which ZTP and its DPs were well separated, with a resolution of >1.5. The stress testing of ZTP was carried out under acidic, alkaline, neutral hydrolysis, oxidative, photolytic and thermal stress conditions. ZTP was found to degrade significantly in acidic, photolytic, thermal and oxidative stress conditions and remain stable in basic and neutral conditions. The developed method was validated with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness as per ICH guidelines. This method was also suitable for the assay determination of ZTP in pharmaceutical dosage forms. The DPs were characterized by LC–MS/MS and their fragmentation pathways were proposed.     

Quantitative and qualitative analysis of common peaks in chemical fingerprint of Yuanhu Zhitong tablet by HPLC-DAD–MS/MS

A quality control (QC) strategy for quantitative and qualitative analysis of “common peaks” in chemical fingerprint was proposed to analyze Yuanhu Zhitong tablet (YZT), using high performance liquid chromatography with diode array detector and tandem mass spectrometry (HPLC-DAD–MS/MS). The chromatographic separation was achieved on an Agilent Eclipse plus C₁₈ column with a gradient elution using a mixture of 0.4‰ ammonium acetate aqueous (pH 6.0 adjusted with glacial acetic acid) and acetonitrile. In chemical fingerprint, 40 peaks were assigned as the “common peaks”. For quantification of “common peaks”, the detection wavelength was set at 254 nm, 270 nm, 280 nm and 345 nm, respectively. The method was validated and good results were obtained to simultaneously determine 10 analytes (protopine, jatrorrhizine, coptisine, palmatine, berberine, xanthotoxin, bergapten, tetrahydropalmatine, imperatorin and isoimperatorin). For qualification of “common peaks”, 33 compounds including 10 quantitative analytes were identified or tentatively characterized using LC–MS/MS. These results demonstrated that the present approach may be a powerful and useful tool to tackle the complex quality issue of YZT.     

Simultaneous determination of nortriptyline hydrochloride and fluphenazine hydrochloride in microgram quantities from low dosage forms by liquid chromatography–UV detection

A novel method for the simultaneous high-performance liquid chromatographic determination of nortriptyline hydrochloride and fluphenazine hydrochloride was developed and validated. Fluvastatin sodium was used as internal standard. The determination was performed on a Hypersil Gold C₈ column (250 mm × 4.6 mm i.d., 5 μm particle size) at 25 °C; the mobile phase, consisting of a mixture of formic acid (0.1 M, pH 2.16)-methanol (33:67, v/v), was delivered at a flow rate of 1.1 mL/min and detector wavelength at 251 nm. The retention time of nortriptyline, fluphenazine and fluvastatin was found to be 5.11, 8.05 and 11.38 min, respectively. Linearity ranges were 5.0–1350.0 and 10.0–1350.0 μg/mL with limit of detection values of 0.72 and 0.31 μg/mL, for nortriptyline and fluphenazine, respectively. Results of assay and recovery studies were statistically evaluated for its accuracy and precision. Correlation coefficients (r²) of the regression equations were greater than 0.999 in all cases. According to the validation results, the proposed method was found to be specific, accurate, precise and could be applied to the simultaneous quantitative analysis of nortriptyline and fluphenazine.     

The expression of adrenomedullin in rats adrenal medulla hyperplasia induced by reserpine and its significance

Objective To observe adrenomedullin （ADM） in adrenal tissue and plasma of rats with adrenal medulla hyperplasia induced by reserpine and study its significance.Methods Thirty male Wistar rats were randomly divided into 2 groups：group A,normal control;group B,experimental model of adrenal medulla hyperplasia,     

Detection of IgH and T-cell receptor gene rearrangement in leukemia by PCR amplification and its clinical application

Therearragementoftheigheavychain(IgH)andT--cellreceptor(TCR)genecanserveasmarkersoftheclonalityoflymphoblasticleukemia.Polymerasechainreaction(PCR)--basedmethodprovidesanopportunityforustoexaminetherearrangedgeneswithanimprovementinsensitivityandspecificityovertraditionalmethodsll].InthisreportwediscusstheuseofasimplePCRxbasedapproachbyacombineddetectionoflgH,TCRVYI--JYandTCRV82--D83generearrangementsin45patientswithacutelymphoblasticleukemia(ALL)patientsandasimpledetectionofthelgH…     

Development of a sensitive and rapid method for quantitation of (S)-(−)- and (R)-(+)-metoprolol in human plasma by chiral LC–ESI–MS/MS

A selective, sensitive and high throughput liquid chromatography-tandem mass spectrometry (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 μm) column. Solid phase extraction of (S)-(−)- and (R)-(+)-metoprolol and rac-metoprolol-d6 as an internal standard (IS) was achieved on Lichrosep DVB HL cartridges employing 200 μL 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.     

Profiling the dynamics of abscisic acid and ABA-glucose ester after using the glucosyltransferase UGT71C5 to mediate abscisic acid homeostasis in Arabidopsis thaliana by HPLC–ESI-MS/MS

The HPLC–MS/MS method was developed to profile the dynamics of abscisic acid (ABA) and ABA-glucose ester (ABA-GE) after cloning glycosyltransferase enzyme family gene AtUGT71C5 into Arabidopsis thaliana. By constructing over-expression lines (OE) and down-expression lines (DN), we acquired mutant strains to analyze the function of AtUGT71C5. The multiple-reaction monitoring (MRM) was used for quantitative determination in negative mode. The transition was m/z 263.1→153.0 for ABA ([M–H]⁺), m/z 425.1→263.0 for ABA-GE ([M–H]⁺), and m/z 321.0→152.0 for chloramphenicol. The linear range was 0.8684–217.1 ng/mL for ABA and 0.3920–196.0 ng/mL for ABA-GE. The accuracy was 88.0–109.0% for ABA and 86.6–113.0% for ABA-GE; the inter-day and intra-day precisions were less than 5.4% for ABA and 8.9% for ABA-GE, respectively. This method is simple and sensitive enough for determination of ABA and ABA-GE in A. thaliana leaves. All the evidence confirmed the speculation that AtUGT71C5 can mediate abscisic acid homeostasis.     

Identification,synthesis and characterization of an unknown process related impurity in eslicarbazepine acetate active pharmaceutical ingredient by LC/ESI–IT/MS, 1H, 13C and 1H–1H COSY NMR

A new impurity was detected during high performance liquid chromatographic (HPLC) analysis of eslicarbazepine acetate active pharmaceutical ingredient. The structure of unknown impurity was postulated based on liquid chromatography mass spectrometry using electrospray ionization and ion trap analyzer (LC/ESI–IT/MS) analysis. Proposed structure of impurity was unambiguously confirmed by synthesis followed by characterization using ¹H, ¹³C nuclear magnetic resonance spectrometry (NMR), ¹H–¹H correlation spectroscopy (COSY) and infrared spectroscopy (IR). Based on the spectroscopic and spectrometric data, unknown impurity was characterized as 5-carbamoyl-10,11-dihydro-5H-dibenzo[b,f]azepin-10-yl propionate.     

Simultaneous quantitation of atorvastatin and its two active metabolites in human plasma by liquid chromatography/(–) electrospray tandem mass spectrometry

A sensitive, accurate and selective liquid chromatography–tandem mass spectrometry method (LC–MS/MS) was developed and validated for the simultaneous quantitation of atorvastatin (AT) and its equipotent hydroxyl metabolites, 2-hydroxy atorvastatin (2-AT) and 4-hydroxy atorvastatin (4-AT), in human plasma. Electrospray ionization (ESI) interface in negative ion mode was selected to improve the selectivity and the sensitivity required for this application. Additionally, a solid phase extraction (SPE) step was performed to reduce any ion-suppression and/or enhancement effects. The separation of all compounds was achieved in less than 6 min using a C₁₈ reverse-phase fused-core^® column and a mobile phase, composed of a mixture of 0.005% formic acid in water:acetonitrile:methanol (35:25:40, v/v/v), in isocratic mode at a flow rate of 0.6 mL/min. The method has lower limit of quantitation (LLOQ) of 0.050 ng/mL for all analytes. The method has shown tremendous reproducibility, with intra- and inter-day precision less than 6.6%, and intra- and inter-day accuracy within ±4.3% of nominal values, for all analytes, and has proved to be highly reliable for the analysis of clinical samples.     

Changes of negative charge on RBC and glomeruli and its relation to the proteinuria of adriamycin nephrosis in rats.

The depth and area-percentage of Alcian blue (AB) staining of glomeruli, the ability of RBC to bind AB and the onset of proteinuria in adriamycin nephrosis in rats were studied. The results showed that the negative charge (NC) on RBC and glomeruli decreased significantly after injection of adriamycin and there was a positive correlation between them (r = 0.77, P less than 0.01). There was a negative correlation between the amount of NC on the RBC and the amount of urinary protein (r = -0.5231, P less than 0.01), and also between the amount of NC on the glomeruli and the amount of urinary protein (r = -0.5140, P less than 0.05).

     

Polymorphism of DXS102 locus in Chinese population and its application to gene diagnosis in hemophilia B family.

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Purification of Ricinus communis agglutinin and its application to research on oligosaccharides in serum of patients with lung cancer

In this report,purified Ricinus communis agglutinin (RCAl) was employed for analysis ofoligosaccharide molecules in serum of patients with lung cancer or benign diseases.By meansof DEAE-Sephadex A-50 and Sephadex G-100 chromatography,RCAl was purified from the ex-tracts of castor beans.After characterizing its bioactivity by means of red blood coagglutination andpurification by SDS-PAGE,RCAl was coupled with CNBr-activated Sepharose 4B.By RCAl af-finity chromatography,the gtycoproteins containing Galβl-4GlcNAc-linked unit at the nonreducingend of the carbohydrate chain were isolated from the gastric cancer tissue.By means of RCAl andthis glycoproteins labelled with horseradish peroxidase,a competitive binding assay was establishedand employed to detect the level of the oligosaccharides defined by RCAl in serum of pateins withlung cancer or benign diseases and of normal persons.The results suggest that expression of theseoligosaccharide molecules is higher in serum of patient with lung cancer than in serum of patientwith a benign disease of the respiratory system and healthy individuals.The positive rates were60.6%(20/33) in serum of patients with lung cancer,21.4% (6/28) with benign diseases and 4.0%(1/25) of normal persons.It is confirmed that detection of these oligosaccharide molecules in serumcan be helpful in the study and diagnosis of lung cancer.