Development and validation of a HPLC-ESI-MS/MS method for the determination of 5-aminosalicylic acid and its major metabolite N-acetyl-5-aminosalicylic acid in human plasma

A new HPLC method for the determination of 5-aminosalicylic acid (5-ASA) and N-acetyl-5-aminosalicylic acid (N-Ac-5-ASA) in human plasma was developed and validated. Plasma samples were analyzed after protein precipitation with methanol and the two analytes were separated using a C18 column with a mobile phase composed of 17.5mmol/L acetic acid (pH 3.3):acetonitrile=85:15 (v/v) at 0.2mL/min flow rate. 4-ASA and N-Ac-4-ASA were used as internal standards. Selective detection was performed by tandem mass spectrometry with electrospray source, operating in negative ionization mode and in multiple reaction monitoring acquisition (m/z 152-->108 for 5-ASA; m/z 194-->150 and 194-->107 for N-Ac-5-ASA). The limit of quantification (LOQ) was 50ng/mL for both analytes (0.2ng injected) and matrix-matched standard curves showed linearity up to 4000ng/mL. In the entire analytical range the within- and between-batch precision (R.S.D.%) values were respectively 90% for 5-ASA and >95% for N-Ac-5-ASA (R.S.D.%相似文献   

Development and validation of an LC-MS/MS method for the quantitative determination of aripiprazole and its main metabolite, OPC-14857, in human plasma

An accurate, sensitive, reproducible, and selective liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for determination of aripiprazole and its main metabolite, OPC-14857, in human plasma was developed and validated. Chromatographic separation was achieved isocratically on a C18 reversed-phase column within 7.5 min. The calibration curve, ranging from 0.1 to 100 ng/ml, was fitted to a 1/y2-weighted linear regression model. The assay showed no significant interference. Lower limit of quantitation (LLOQ) for both analytes was 0.1 ng/ml using 0.4 ml of plasma. Intra- and inter-assay precision and accuracy values for aripiprazole and OPC-14857 were within regulatory limits.     

Development and validation of a sensitive LC/MS/MS method for the simultaneous determination of naloxone and its metabolites in mouse plasma

A rapid, specific, and reliable LC-MS/MS based bioanalytical method was developed and validated for the simultaneous determination of naloxone (NLX) and its two metabolites, 6β-naloxol (NLL) and naloxone-3β-D-glucuronide (NLG) in mouse plasma. The optimal chromatographic behavior of these analytes was achieved on an Aquasil C18 column (50 mm × 2.1 mm, 5 μm) using reversed phase chromatography. The total LC analysis time per injection was 2.5 min with a flow rate of 1.0 mL/min with gradient elution. Sample preparation via protein precipitation with acetonitrile in a 96-well format was applied for analyses of these analytes. The analytes were monitored by electrospray ionization in positive ion multiple reaction monitoring (MRM) mode. Modification of collision energy besides chromatographic separation was applied to further eliminate interference peaks for NLL and NLG. The method validation was conducted over the curve range of 0.200/0.400/0.500 to 100/200/250 ng/mL for NLX/NLL/NLG, respectively, using 0.0250 mL of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels showed ≤ 6.5% relative standard deviation (RSD) and -8.3 to -2.5% relative error (RE). The method was successfully applied to determine the concentrations of NLX, NLL, and NLG in incurred mouse plasma samples.     

Development and validation of a LC-MS/MS method for the determination of viaminate in human plasma

A sensitive and specific method for determination of viaminate in human plasma by using high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (LC-MS/MS) was developed in this study. The plasma samples were simply deproteinated, extracted, evaporated, and then reconstituted in 200 microl of methanol prior to analysis. Chromatographic separation was carried out on a Shimadzu VP-ODS column (250 mm x 2.0 mm, 5 microm) with a mobile phase of methanol-water (95:5, v/v) at a flow rate of 0.2 ml/min. Quantification was performed in the negative-ion electrospray ionization mode by selected ion monitoring of the product ions at m/z 164 for viaminate and m/z 109 for testosterone propionate which was used as the internal standard. The corresponding parent ions were m/z 446 and m/z 345. A linear calibration curve was observed within the concentration range of 0.10-200 ng/ml. The lowest limit of quantitation (LLOQ) was 0.1 ng/ml. The extraction-efficiency at three concentrations was 100.7, 93.6, and 99.7%. Practical utility of this new LC-MS/MS method was confirmed in pilot pharmacokinetic studies in humans following oral administration.     

Development and validation of a LC-ESI-MS/MS method for the determination of swertiamarin in rat plasma and its application in pharmacokinetics

A new LC-ESI-MS/MS assay method has been developed and validated for the quantification of swertiamarin, a representative bioactive substance of Swertia plants, in rat plasma using gentiopicroside, an analog of swertiamarin on chemical structure and chromatographic action, as the internal standard (IS). The swertiamarin and IS were extracted from rat plasma using solid-phase extraction (SPE) as the sample clean-up procedure, and they were chromatographed on a narrow internal diameter column (Agilent ZORBAX ECLIPSE XDB-C(18) 100 mm × 2.1 mm, 1.8 μm) with the mobile phase consisting of methanol and water containing 0.1% acetic acid (25:75, v/v) at a flow rate of 0.2 mL/min. The detection was performed on an Agilent G6410B tandem mass spectrometer by negative ion electrospray ionisation in multiple-reaction monitoring mode while monitoring the transitions of m/z 433 [M+CH(3)COO](-)→179 and m/z 415 [M+CH(3)COO](-)→179 for swertiamarin and IS, respectively. The lower limit of quantification (LLOQ) was 5 ng/mL within a linear range of 5-1000 ng/mL (n=7, r(2)≥0.994), and the limit of detection (LOD) was demonstrated as 1.25 ng/mL (S/N≥3). The method also afforded satisfactory results in terms of sensitivity, specificity, precision (intra- and inter-day), accuracy, recovery, freeze/thaw, long-time stability and dilution integrity. This method was successfully applied to determination of the pharmacokinetic properties of swertiamarin in rats after oral administration at a dose of 20 mg/kg. The following pharmacokinetic parameters were obtained (mean): maximum plasma concentration, 1920.1 ng/mL; time to reach maximum plasma concentration, 0.945 h; elimination half-time, 1.10h; apparent total clearance, 5.638 L/h/kg; and apparent volume of distribution, 9.637 L/kg.     

Development and validation of an LC-MS/MS method for determination of 8-iso-prostaglandin f2 Alpha in human saliva

BackgroundIncreased formation of reactive oxygen species may be caused by the ion release of the metal alloys used in prosthetic dental restorations due to the corrosion process. As products of lipid peroxidation, isoprostanes can be used as a marker for oxidative stress in the body. There are two significant advantages of using isoprostanes as an oxidative stress marker - presence in all fluids in the body and low reactivity. Saliva provides noninvasive, painless, and cost-effective sample collection and can be used as an alternative testing medium of blood and urine.MethodsThis study presents the development and validation of a sample LC-MS/MS method to quantify 8-isoprostaglandin F2-a in human saliva using salt-out assisted liquid-liquid extraction (SALLE).ResultsThe selected sample preparation procedure optimized chromatographic separation and mass detection provided high recovery and sensitivity of the analysis. The calibration curve was obtained in the predefined range 25-329 ng/L with R2 larger than 0.995. Normalized matrix varied between 89.7 % and 113.5%. The method showed sufficient accuracy and precision - accuracy in the range 89.7 %-113.9 %, and precision between 2.3% and 5.4%.ConclusionsThe proposed method is validated according to current EMA/FDA industrial guidance for bioanalysis and offers an appropriate level of sensitivity and sufficient accuracy and precision.     

Development and validation of an LC/MS/MS assay for mycophenolic acid in human peripheral blood mononuclear cells

The aim was to develop a LC/MS/MS method able to quantify mycophenolic acid (MPA) in the peripheral blood mononuclear cells (PBMCs) of transplanted patients. PBMCs were isolated from blood by a density gradient separation. The chromatographic separation was carried out on a Zorbax Stable Bond CN, 150 mmx2.1 mm, and MS/MS detection was performed after positive electrospray ionisation of the protonated parent ion. The calibration range was from 0.25 to 100 ng/sample. Extraction from the cells and ionisation recoveries reached 73.5 and 37.9%, respectively. Inaccuracy was always <10% with CVs<15%. MPA was stable at room temperature in the autosampler over 48 h and at -20 degrees C over 1.5 months. Application to clinical samples taken from patients treated with mycophenolate mofetil indicated that the method is suitable for measuring intracellular MPA.     

Quantitative thin-layer chromatographic method for the determination of procainamide and its major metabolite in plasma

A sensitive and accurate spectrodensitometric method was developed for the determination of procainamide and its major metabolite, N-acetylprocainamide, in plasma. The method involves extraction into organic solvent at alkaline pH, separation by thin-layer chromatography and direct measurement of the adsorbance of the compounds on the plate at 275 nm. Quantities as low as 10 ng could be measured and a linear relationship was obtained between peak areas and amounts of the compounds in the spots from 10 to 200 ng. The recovery of both drugs from plasma was from 95.4 to 104.8%. The method is sensitive and specific, and procainamide was well separated from N-acetylprocainamide at all investigated concentrations. The method is recommended for clinical assays and pharmacokinetics studies.     

Development and validation of a sensitive HPLC-ESI-MS/MS method for the direct determination of glucosamine in human plasma

A sensitive and specific HPLC-ESI-MS/MS method for the direct determination of glucosamine in human plasma has been developed and validated. Plasma samples were analyzed after a simple, one-step protein precipitation clean-up with trichloroacetic acid using a polymer-based amino high-performance liquid chromatography (HPLC) column and a water/acetonitrile mobile phase elution gradient, with d-[1-(13)C]glucosamine as the internal standard. Detection was performed by mass spectrometry, using an electrospray source and employing multiple reaction monitoring to separately monitor glucosamine and the internal standard. The limit of quantification of the method was 10ng/ml of glucosamine and the calibration curve showed a good linearity up to 1000ng/ml. The precision (R.S.D.) and the accuracy (bias) of the method at the limit of quantification were 13.8 and 4.0%, respectively, and the mean recovery of glucosamine at three concentration levels was 101.6+/-5.7%. The method was applied for the determination of glucosamine concentrations in human plasma samples collected from untreated healthy volunteers and, in a separate bioavailability study, to evaluate plasma glucosamine pharmacokinetics profiles after oral administration of crystalline glucosamine sulfate.     

Development and validation of a LC/MS/MS method for simultaneous quantification of oxcarbazepine and its main metabolites in human serum

A fast, sensitive and specific LC/MS/MS method for the simultaneous analysis of oxcarbazepine (OXC), 10-hydroxycarbazepine (MHD) and trans-diol-carbazepine (DHD), in human serum, has been developed and validated. Serum drugs were extracted by C8 solid-phase cartridges (SPE) and separated in less than 3 min on a C18 reverse-phase column using an isocratic elution. A tandem mass spectrometer, as detector, was used for quantitative analysis in positive mode by a multiple reaction monitoring. Calibration curves, obtained on two ranges of concentration (0.78-50 mg/L for MHD and 0.078-5.0 mg/L for OXC and DHD), showed correlation coefficients (r) better than 0.997. Within day and between days quality controls imprecision, as CV%, ranged from 0.3 to 4.6% and from 1.9 to 5.8%, respectively. Cyheptamide (CYE) was used as internal standard. No detectable carry-over and no relevant cross-talk and matrix effect occurred. Samples from 24 treated patients were analysed and drug serum concentrations obtained by this method are in agreement with those of other methods and also are well correlated (r=0.88) in comparison to our routine HPLC-UV method. Based on the analytical results and short run time, the method is suitable to support routine analysis of therapeutic drugs monitoring from human serum of treated patients or for pharmacokinetic studies.     

Development and validation of a LC/MS/MS method for quantifying the next generation calcineurin inhibitor, voclosporin, in human whole blood

A rapid, accurate, and reproducible liquid chromatography electrospray tandem mass spectrometry (LC/ESI-MS/MS) method was developed and validated for the therapeutic drug monitoring of voclosporin in human whole blood. Sample aliquots of 100muL were processed utilizing a protein precipitation procedure that contained a mixture of methanol, 0.2M ZnSO(4), and deuterated voclosporin internal standard. Supernatant was injected onto a Zorbax SB-C8, 2.1x12.5mm column (at 60 degrees C), and washed with water-acetonitrile, supplemented with 0.02% glacial acetic acid and 0.02mM sodium acetate, to remove poorly retained components. After washing, water-MeOH (with 0.02% glacial acetic acid and 0.02mM sodium acetate) was used to elute the voclosporin and internal standard to the Applied Biosystems/MDS-Sciex API3000 mass spectrometer for detection in multiple reaction monitoring. Analytical performance was assessed in the range of 1-200ng/ml in whole blood. This method has been used to quantify concentrations of voclosporin in whole blood from healthy volunteers participating in a pharmacokinetic study.     

Development and validation of liquid chromatography-tandem mass spectrometric method for simultaneous determination of fosinopril and its active metabolite fosinoprilat in human plasma

A highly sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous determination of the prodrug fosinopril and its major active metabolite fosinoprilat for pharmacokinetic studies in healthy subjects. In order to get the lower limit of quantification (LLOQ), especially for analysis of fosinopril, key points of the method have been investigated including chromatographic conditions and selection of LC-MS/MS conditions. The analytes were extracted from plasma samples by liquid-liquid extraction, separated on a reversed-phase C(8) column using gradient procedure, and detected by tandem mass spectrometry with a triple quadrupole ionization interface. The analytes and internal standard zaleplon were detected using positive electrospray ionization (ESI) in the SRM mode. The LLOQ of the method down to 0.1 ng mL(-1) for fosinopril and 1.0 ng mL(-1) for fosinoprilat were identifiable and reproducible. The standard calibration curves for both fosinopril and fosinoprilat were linear over the ranges of 0.1-15.0 and 1.0-700 ng mL(-1) in human plasma, respectively. The within- and between-batch precisions (relative standard deviation (RSD)%) and the accuracy were acceptable. The validated method was successfully applied to reveal the pharmacokinetic properties of fosinopril and fosinoprilat after oral administration.     

Development and validation of an HPLC method for the determination of spironolactone and its metabolites in paediatric plasma samples

An HPLC method has been developed and validated for the determination of spironolactone, 7 alpha-thiomethylspirolactone and canrenone in paediatric plasma samples. The method utilises 200 microl of plasma and sample preparation involves protein precipitation followed by Solid Phase Extraction (SPE). Determination of standard curves of peak height ratio (PHR) against concentration was performed by weighted least squares linear regression using a weighting factor of 1/concentration2. The developed method was found to be linear over concentration ranges of 30-1000 ng/ml for spironolactone and 25-1000 ng/ml for 7 alpha-thiomethylspirolactone and canrenone. The lower limit of quantification for spironolactone, 7 alpha-thiomethylspirolactone and canrenone were calculated as 28, 20 and 25 ng/ml, respectively. The method was shown to be applicable to the determination of spironolactone, 7 alpha-thiomethylspirolactone and canrenone in paediatric plasma samples and also plasma from healthy human volunteers.     

Development and validation of a spectral library searching method for peptide identification from MS/MS

A notable inefficiency of shotgun proteomics experiments is the repeated rediscovery of the same identifiable peptides by sequence database searching methods, which often are time-consuming and error-prone. A more precise and efficient method, in which previously observed and identified peptide MS/MS spectra are catalogued and condensed into searchable spectral libraries to allow new identifications by spectral matching, is seen as a promising alternative. To that end, an open-source, functionally complete, high-throughput and readily extensible MS/MS spectral searching tool, SpectraST, was developed. A high-quality spectral library was constructed by combining the high-confidence identifications of millions of spectra taken from various data repositories and searched using four sequence search engines. The resulting library consists of over 30,000 spectra for Saccharomyces cerevisiae. Using this library, SpectraST vastly outperforms the sequence search engine SEQUEST in terms of speed and the ability to discriminate good and bad hits. A unique advantage of SpectraST is its full integration into the popular Trans Proteomic Pipeline suite of software, which facilitates user adoption and provides important functionalities such as peptide and protein probability assignment, quantification, and data visualization. This method of spectral library searching is especially suited for targeted proteomics applications, offering superior performance to traditional sequence searching.     

Development of an HPLC-MS/MS method for the selective determination of paracetamol metabolites in mouse urine

An HPLC-MS/MS method has been developed for the selective quantitative analysis of paracetamol and its two major metabolites. The use of tandem MS enabled the detection and quantitation of metabolites in small sample sizes with high sensitivity and selectivity. Isocratic elution using acetonitrile and water containing formic acid combined with electrospray-tandem MS enabled the separation and accurate quantitation of each analyte and the internal standard 3-acetamidophenol. The on-column limits of detection for paracetamol, paracetamol sulfate, and paracetamol glucuronide were 2.4, 1.2, and 1.2 pmol, respectively. The method was applied to quantitate paracetamol and its metabolites in mouse urine. It is highly specific, sensitive, and easily adaptable to measure these analytes in biological fluids of other animals.     

Development and validation of a sensitive ultra performance liquid chromatography tandem mass spectrometry method for the analysis of fentanyl and its major metabolite norfentanyl in urine and whole blood in forensic context

Fentanyl and its major metabolite norfentanyl often occur in low doses in biological samples. Therefore, a highly sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been developed and fully validated. Sample preparation was performed on a mixed-mode cation exchange solid phase extraction (SPE) cartridge with an additional alkaline wash step to decrease matrix effects and thus increase sensitivity. Ionization of fentanyl and norfentanyl with electrospray ionization (ESI) was more efficient than atmospheric pressure chemical ionization (APCI). The use of a mobile phase of high pH resulted in higher ESI signals than the conventional low pH mobile phases. In the final method, gradient elution with 10 mM ammonium bicarbonate (pH 9) and methanol was performed. A comparison of columns with different internal diameter and/or smaller particles showed optimal resolution and sensitivity when an Acquity C18 column (1.7 μm, 2.1 mm × 50 mm) was used. Deuterium labeled internal standards were used, but with careful evaluation of their stability since loss of deuteriums was seen. With limits of detection of 0.25 pg/ml for fentanyl and 2.5 pg/ml for norfentanyl in urine and 5 pg/ml for fentanyl and norfentanyl in whole blood the presented method is highly appropriate for the analysis of fentanyl and norfentanyl in forensic urine and blood samples.     

A sensitive assay for simultaneous determination of plasma concentrations of valganciclovir and its active metabolite ganciclovir by LC/MS/MS

A protein precipitation, liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the simultaneous determination of valganciclovir and its active metabolite ganciclovir in human plasma. The solvent system also served as a protein precipitation reagent. The chromatographic separation was achieved on an Aquasil C18 column (50 mm x 2.1mm, 5 microm). A linear gradient mobile phase between 0.02% formic acid and methanol was used. Detection was by positive ion electrospray tandem mass spectrometry on a Sciex API3000. The standard curves, which ranged from 4 to 512 ng/mL for valganciclovir and from 0.1 to 12.8 microg/mL for ganciclovir, were fitted to a 1/x weighted quadratic regression model. The method was proved to be accurate, specific and sensitive enough and was successfully applied to a pharmacokinetic study.     

Development and validation of an HPLC-UV method for determination of iohexol in human plasma

An HPLC-UV analytical method for estimation of iohexol in human plasma was developed and validated. Protein precipitation and iohexol extraction from plasma (100 microl) was carried out by adding 800 microl perchloric acid (5%, v/v in water) containing iohexol related compound B as the internal standard followed by vortex mixing and centrifugation. The supernatant (90 microl) was then injected onto a microBondapak C(18) column (150 mm x 3.9 mm, 10 microm) maintained at 30 degrees C. The mobile phase comprised of various proportions of acetonitrile and water with a total run time of 12 min and the wavelength of the UV detector was set at 254 nm. The extraction recovery of iohexol from plasma was >95% and the calibration curve was linear (r(2)=0.99) over iohexol concentrations ranging from 10 to 750 microg/ml (n=8). The method had an accuracy of >92% and intra- and inter-day CV of <3.7% and <3.6%, respectively. The method reported is simple, reliable, precise, accurate and has the capability of being used for determination of iohexol in clinical settings.     

LC-MS/MS method for simultaneous determination of valproic acid and major metabolites in human plasma

A rapid and sensitive method using liquid chromatography-tandem mass spectroscopy (LC-MS/MS) was developed and validated for simultaneous quantitative determination of valproic acid and three major metabolites (3-OH-valproic acid, 4-ene-valproic acid and 5-OH-valproic acid) in human plasma. The analytes and internal standard were isolated from 200 μL samples by solid phase extraction using a ZORBAX SB-C? column (3.5 μm, 2.1×100 mm) with an isocratic mobile phase consisting of methanol-10mM ammonium acetate (80:20, v/v) containing 0.1% formic acid at a flow rate of 0.3 mL/min. The method had a chromatographic total run time of 2.0 min. The lower limit of quantification of valproic acid, 3-OH-valproic acid, 4-ene-valproic acid and 5-OH-valproic acid of the method was 2030, 51.5, 50.15 and 51.25 ng/mL, respectively. The method was linear for valproic acid and the three metabolites with correlation coefficients >0.995 for all analytes. The intra-day and inter-day accuracy and precision of the assay were less than 15.0%. This analytical method was successfully used to assay plasma concentrations of valproic acid and the three metabolites in human plasma from epileptic patients.