Chromatography-positive Electrospray Tandem Mass Research Articles

First Evidence of the Cysteine and Glutathione Conjugates of 3-Sulfanylpentan-1-ol in Hop ( Humulus lupulus L.).

After evidence of the cysteinylated precursors of 3-sulfanyl-4-methylpentan-1-ol (Cys-26) and 3-sulfanylhexan-1-ol (Cys-23) in hop, S-glutathione precursors (G-23 and G-26) were recently discovered in different dual-purpose hop varieties. Because free 3-sulfanylpentan-1-ol (21) has also been detected in hop, the present work aimed to identify its potential precursors. The compounds S-3-(1-hydroxylpentyl)cysteine (Cys-21) and S-3-(1-hydroxylpentyl)glutathione (G-21) were first synthesized and characterized by nuclear magnetic resonance and high-resolution mass spectrometry. High-performance liquid chromatography-positive electrospray ionization-tandem mass spectrometry evidenced both for the first time in hop. Both S conjugates were further quantitated in six hop samples: the well-known Saaz, Amarillo, Citra, Hallertau Blanc, Nelson Sauvin, and Polaris. Similar to G-23, G-21 appeared ubiquitous to all varieties. Of all of the samples investigated here, Citra (harvest 2017) emerged as the richest in G-21, with 18 mg/kg of dry matter. Cys-21 was found in all samples at a much lower concentration (up to 0.2 mg/kg of dry matter in Polaris, harvest 2017). Model media spiked with Cys-21 or G-21 allowed for the confirmation that brewing yeast is able to release free compound 21 from them.

Phytohormone metabolism in human cells: Cytokinins are taken up and interconverted in HeLa cell culture.

Cytokinins (CKs) encompass a group of phytohormones, known to orchestrate many critical processes in plant development. Excluding Archaea, CKs are pervasive among all kingdoms, but much less is reported about their metabolism beyond plants. Recent evidence from mammalian tissues indicates the presence of six additional CK forms beyond the previously identified, single mammalian CK, N6‐isopentenyladenosine (i6A). There is limited understanding of CK biosynthesis pathways in mammalian systems; therefore, human cervical cancer (HeLa) cells were used to further characterize CK processing by tracking the interconversion of CKs into their various structural derivatives in mammalian cells in a time‐course study. Through high‐performance liquid chromatography‐positive electrospray ionization‐tandem mass spectrometry (HPLC‐(+ESI)‐MS/MS), we document changes in the functional profiles of endogenous CKs in a human cell line following metabolism by HeLa cell cultures. The nucleotide CK fraction (iPRP) was found exclusively within the cell pellet (0.34 pmol/106 cells), and the active free base (FB) form (iP) and riboside fraction (iPR) were found in greater abundance extracellularly (1.67 and 0.10 nmol/L respectively). For further confirmation, we demonstrate that HeLa cells metabolize an exogenously supplied CK, N6‐benzyladenosine (BAR). In the HeLa culture supernatant, a 12‐fold decrease in BAR concentration was observed within the first 24 hours of incubation accompanied by a fivefold increase in the FB form, N6‐benzyladenine (BA). These findings support the hypothesis that HeLa cells have the enzymatic pathways required for the metabolism of both endogenous and exogenous CKs.

Quantitative determination of 13 organophosphorous flame retardants and plasticizers in a wastewater treatment system by high performance liquid chromatography tandem mass spectrometry

A method for quantitative determination of 13 organophosphorous compounds (OPs) was developed and applied to influent, primary sludge, activated sludge, biosolids, primary effluent and final effluent from a wastewater treatment plant (WWTP). The method involved solvent extraction followed by solid phase clean-up and analysis by high performance liquid chromatography positive electrospray ionization-tandem mass spectrometry (HPLC(+ESI)MS/MS). Replicate spike/recovery experiments revealed the method to have good accuracy (70–132%) and precision (<19% RSD) in all matrices. Detection limits of 0.1–5ng/L for aqueous samples and 0.01–0.5ng/g for solid samples were achieved. In the liquid waste stream ∑OP concentrations were highest in influent (5764ng/L) followed by primary effluent (4642ng/L), and final effluent (2328ng/L). In the solid waste stream, the highest ∑OP concentrations were observed in biosolids (3167ng/g dw), followed by waste activated sludge (2294ng/g dw), and primary sludge (2128ng/g dw). These concentrations are nearly 30-fold higher than ∑polybrominated diphenyl ether (BDE) concentrations in influents and nearly 200-fold higher than ∑BDE concentrations in effluents from other sites in Canada. Tetrekis(2-chlorethyl)dichloroisopentyldiphosphate (V6), tripropylphosphate (TnPrP), and Tris(2,3-dibromopropyl)phosphate (TDBPP) are investigated for the first time in a WWTP. While TnPrP and TDBB were not detected, V6 was observed at concentrations up to 7.9ng/g in solid waste streams and up to 40.7ng/L in liquid waste streams. The lack of removal of OPs during wastewater treatment is a concern due to their release into the aquatic environment.

Development and validation of an ultra high performance liquid chromatography tandem mass spectrometry method for simultaneous determination of sulfonamides, quinolones and benzimidazoles in bovine milk.

A simple, sensitive and reliable analytical method was developed for the simultaneous determination of 38 veterinary drugs (18 sulfonamides, 11 quinolones and 9 benzimidazoles) and 8 metabolites of benzimidazoles in bovine milk by ultra high performance liquid chromatography-positive electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS). Samples were extracted with acidified acetonitrile, cleaned up with Oasis(®) MCX cartridges, and analyzed by LC-MS/MS on an Acquity UPLC(®) BEH C18 column with gradient elution. The method allows such multi-analyte measurements within a 13min runtime while the specificity is ensured through the MRM acquisition mode. The method was validated according to the European Commission Decision 2002/657/EC determining specificity, decision limit (CCα), detection capability (CCβ), recovery, precision, linearity and stability. For compounds which have MRLs in bovine milk, the CCα values fall into a range from 11 to 115μg/kg, and the CCβ values fall within a range of 12-125μg/kg. For compounds which have not MRLs in bovine milk, the CCα values fall into a range from 0.01 to 0.08μg/kg, and the CCβ values fall within a range of 0.02-0.11μg/kg. The mean recoveries of the 46 analytes were between 87 and 119%. The calculated RSD values of repeatability and within-laboratory reproducibility experiments were below 11% and 15% for the 46 compounds, respectively. The method was demonstrated to be suitable for the simultaneous determination of sulfonamides, quinolones and benzimidazoles in bovine milk.

Screening of Danish traffic cases for synthetic cannabinoids in whole blood by LC-MS/MS

ABSTRACT A target screening method for the detection of 13 synthetic cannabinoids in whole blood was developed and validated. Samples underwent automated solid-phase extraction, and sample extracts were analyzed by liquid chromatography-positive electrospray ionization-tandem mass spectrometry using two transitions in multiple reaction monitoring mode. The limit of detection was between 0.1-2.5 ng/mL for the compounds except HU-210, and extraction recovery ranged from 59 to 78%. The method was used to screen 393 Danish traffic cases from 2012, where the driver was suspected of driving under the influence of drugs. No synthetic cannabinoids were identified in these samples. Additionally, the method was applied to a clinical intoxication case, and the synthetic cannabinoid AM- 2201 was identified in serum. We conclude that the prevalence of driving under the influence of synthetic cannabinoids in Denmark is likely to be low, and that synthetic cannabinoids are most likely to be encountered in the clinical setting.

SIMULTANEOUS DETERMINATION OF OLANZAPINE AND FLUOXETINE IN HUMAN PLASMA BY LC-MS/MS AND ITS APPLICATION TO PHARMACOKINETIC STUDY

A selective, sensitive, liquid chromatography–positive electrospray ionization tandem mass spectrometry method was developed and validated for the simultaneous quantification of fluoxetine and olanzapine in human plasma using duloxetine (IS) as an internal standard. The analytes and IS were extracted from a 0.250-mL aliquot of human plasma by liquid–liquid extraction using methyl tertiary butyl ether and n-hexane (80:20). The eluted samples were chromatographed with a run time of 3.20 min on X-terra RP8 4.6 × 50 mm, 5 µm column by using a 90:10 v/v mixture of acetonitrile and 5 mM ammonium acetate buffer (P H 5.00 ± 0.05) as an isocratic mobile phase at a flow rate of 0.4 mL/min, and analyzed by mass spectrometry in the multiple reaction monitoring mode using the respective [M+H] + ions, m/z 310.16 → 114.12 for fluoxetine, 313.19 → 256.12 for olanzapine, and 298.14→154.09 for the IS, respectively. Calibration plots were linear over the concentration range of 0.050 to 25.044 ng/mL for fluoxetine and 0.100 to 50.000 ng/mL for olanzapine. Intra-day and inter-day precision and accuracy for quality control samples of fluoxetine (0.150, 12.832, and 19.433 ng/mL) ranged between 2.95 to 10.02% and between 97.90 to 103.25% and for olanzapine (0.300, 25.000, and 38.515 ng/mL) ranged between 3.30 to 7.66% and between 100.75 to 103.11%, respectively. Matrix effect, recovery, and process efficiency experiment was conducted at five different standards, the absolute extraction efficiency was 83.32% for fluoxetine, 86.30% for olanzapine, and 77.73% for IS. This method was successfully applied on pharmacokinetic study.

Validation of an electrospray ionisation LC–MS/MS method for quantitative analysis of telaprevir and its R-diastereomer

A sensitive high-performance reverse phase liquid chromatography-positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of telaprevir and its inactive R-diastereomer (VRT-127394) in human plasma. The analytes and the internal standard (telaprevir-d11) were extracted from plasma by liquid-liquid extraction using tert-Butyl methyl ether (TBME). Chromatographic separation was achieved on a reversed-phase Accucore C18 column with a gradient programme consisting of water:ammonia (25%), 100:0.01 (v/v) (mobile phase A) and ACN:MeOH:ammonia (25%), 15:85:0.01 (v/v/v) (mobile phase B). The MS acquisition was performed with selective reaction monitoring mode using the respective [M+H](+) ions, m/z 680.59→322.42 for telaprevir and VRT-127394, and 691.15→110.13 for telaprevir-d11. The assay exhibited a linear dynamic range of 5-5000ng/mL for telaprevir and VRT-127394. Acceptable precision (%RSD<6.5%) and accuracy (94-108%) were obtained for concentrations over the range of the standard curve. A procedure was established to stabilise the plasma to prevent ex vivo interconversion of the isomers.

Development and validation of an ultra high performance liquid chromatography tandem mass spectrometry method for determination of 10 cephalosporins and desacetylcefapirin in milk

A simple, sensitive and reliable analytical method was developed for the simultaneous determination of 10 cephalosporins and desacetylcefapirin in bovine milk by ultra high performance liquid chromatography-positive electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS). Samples were directly purified through HLB cartridge after dilution with 50mM phosphate buffer solution (pH 8.5). Then the eluate was dried under nitrogen and the residue was redissolved in mobile phase. Samples were analyzed by LC-MS/MS on an Acquity UPLC BEH Shield RP18 column with gradient elution. The samples were quantified using ceftiofur-D3 as internal standard. The proposed method was validated according to the European Commission Decision 2002/657/EC. The CCα values were 111, 0.04, 140, 55, 55, 67, 23, 23, 68, 0.10 and 113μg/kg for cefalexin, cefradine, cefacetrile, cefazolin, cefoperazone, cefapirin, cefalonium, cefquinome, desacetylcefapirin, cefotaxime and ceftiofur, respectively. The mean recoveries, repeatability (expressed as coefficient of variation, CVr), and reproducibility (CVR) varied from 94.6% to 117.1%, from 5.6% to 13.6% (CVr), and from 5.9% to 27.9% (CVR), respectively. The method is demonstrated to be suitable for the determination of 10 cephalosporins and desacetylcefapirin in bovine milk. The total time required for the analysis of one sample, including sample preparation, was about 40min.

Determination of 51 carbamate pesticide residues in vegetables by liquid chromatography-tandem mass spectrometry based on optimization of QuEChERS sample preparation method

The raw extracts of six vegetables (tomato, green bean, shallot, broccoli, ginger and carrot) were analyzed using gas chromatography-mass spectrometry (GC-MS) in full scan mode combined with NIST library search to confirm main matrix compounds. The effects of cleanup and adsorption mechanisms of primary secondary amine (PSA) , octadecylsilane (C18) and PSA + C18 on co-extractives were studied by the weight of evaporation residue for extracts before and after cleanup. The suitability of the two versions of QuEChERS method for sample preparation was evaluated for the extraction of 51 carbamate pesticides in the six vegetables. One of the QuEChERS methods was the original un-buffered method published in 2003, and the other was AOAC Official Method 2007.01 using acetate buffer. As a result, the best effects were obtained from using the combination of C18 and PSA for extract cleanup in vegetables. The acetate-buffered version was suitable for the determination of all pesticides except dioxacarb. Un-buffered QuEChERS method gave satisfactory results for determining dioxacarb. Based on these results, the suitable QuEChERS sample preparation method and liquid chromatography-positive electrospray ionization-tandem mass spectrometry under the optimized conditions were applied to determine the 51 carbamate pesticide residues in six vegetables. The analytes were quantified by matrix-matched standard solution. The recoveries at three levels of 10, 20 and 100 microg/kg spiked in six vegetables ranged from 58.4% to 126% with the relative standard deviations of 3.3%-26%. The limits of quantification (LOQ, S/N > or = 10) were 0.2-10 microg/kg except that the LOQs of cartap and thiofanox were 50 microg/kg. The method is highly efficient, sensitive and suitable for monitoring the 51 carbamate pesticide residues in vegetables.

Simultaneous determination of azathioprine and its metabolite 6-mercaptopurine in human plasma using solid phase extraction-evaporation and liquid chromatography–positive electrospray tandem mass spectrometry

A simple, rapid, specific, sensitive and liquid chromatography coupled with tandem mass spectrophotometric method was developed and validated for the estimation of azathioprine and its metabolite 6-mercaptopurine in human plasma by using lamivudine and 6-mercaptopurine D3 as the internal standard. Azathioprine and 6-mercaptopurine were extracted from human plasma by solid-phase extraction (SPE)-Evaporation method, using Oasis MCX cartridge for cleaning procedure. The stationary phase was chromatographed on a ZORBAX SB CN, (75X50 mm, 5 µ) column where as mobile phase constitutes of acetonitrile: 2mM ammonium acetate (70:30 v/v) at a flow rate of 0.800 ml/min. The detection was performed with an Applied Biosystems Sciex API 4000 mass spectrometer by multiple reaction monitoring (MRM). The method validation proofs were carried out as per the USFDA guidelines as described, showing a linearity system (r2 &gt; 0.99) over a range of 2.455 ng/mL to 106.568 ng/mL for azathioprine and 1.165 ng/mL to 101.143 ng/mL concentrations for 6-mercaptopurine and a recovery shows 99.36% and 100.44% for azathioprine and 6-mercaptopurine respectively. The results show that this proposed approach is effective and can be applied to the extraction and analysis of other pharmaceutical compounds.DOI: http://dx.doi.org/10.3329/icpj.v1i11.12059 International Current Pharmaceutical Journal 2012, 1(11): 342-352

Development and Validation of an LC–ESI-MS Method for Quantitative Determination of Aripiprazole in Human Plasma and an Application to Pharmacokinetic Study

A selective, sensitive, high pressure liquid chromatography-positive electrospray ionization tandem mass spectrometry method was developed and validated for the quantification of aripiprazole in human K(2)EDTA plasma using zolpidem tartrate as an internal standard. The analyte and internal standard were extracted from human plasma by solid-phase extraction using methanol. The eluted samples were chromatographed on a Grace Smart RP 18 4.6 × 100 mm, 3 µ column by using a 95:5 v/v mixture of methanol and ammonium acetate buffer (30 mM, pH 5.0 ± 0.05) as a gradient mobile phase at a flow rate of 0.6 mL/min, and analyzed by mass spectrometry in the multiple reaction monitoring mode using the [M + H](+) ions m/z 448.03 → 285.14 for aripiprazole and m/z 308.13 → 235.25 for the internal standard (zolpidem tartrate), respectively. Calibration plots were linear over the concentration range of 0.20 to 60.01 ng/mL. Intra-day and inter-day precision (percent coefficient of variation) and accuracy (percent nominal) for quality control samples (0.60, 30.60 and 45.59 ng/mL) ranged between 2.28 and 8.93% and between 92.50 and 107.07%, respectively. Extraction recovery of aripiprazole from plasma was in the range 75.56-79.57%; mean recovery was 77.35%. The main pharmacokinetic parameters were T(max) = (4.00 ± 2.336) C(max) = (55.16 ± 13.490) and AUC = (1846.28 ± 484.686).

Determination of phenylethanolamine A in animal hair, tissues and feeds by reversed phase liquid chromatography tandem mass spectrometry with QuEChERS

A simple, sensitive and reliable analytical method was developed for the determination of a new beta-agonist phenylethanolamine A in animal hair, tissues and animal feeds by ultra high performance liquid chromatography–positive electrospray ionization tandem mass spectrometry (UHPLC–ESI-MS/MS) with QuEChERS. Samples were extracted with acetonitrile/water (80:20, v/v). The extract was purified through QuEChERS method, then was dried with nitrogen and residues were redissolved in mobile phase for hair sample or directly diluted with 0.1% formic acid in water for other samples, and analyzed by LC–MS/MS on a Waters Acquity BEH C18 column with 0.1% formic acid in water/methanol as mobile phase with gradient elution. The samples were quantified using phenylethanolamine A-D3 as internal standards. The proposed method was validated according to the European Commission Decision 2002/657/EC determining specificity, decision limit (CCα), detection capability (CCβ), recovery, precision, linearity, robustness and stability. The CCα values ranged from 0.10 to 0.26μg/kg. The CCβ values ranged from 0.20 to 0.37μg/kg. The mean recoveries of 95.4–108.9% with intra-day CVs of 2.2–5.6% and inter-day CVs of 3.1–6.2% were obtained. The method is demonstrated to be suitable for the determination of phenylethanolamine A in animal hair, tissues and animal feeds. The total time required for the analysis of one sample except animal hair sample, including sample preparation, was about 25min.

LC‐ESI‐MS/MS method for quantification of ambrisentan in plasma and application to rat pharmacokinetic study

A sensitive high-performance liquid chromatography-positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of ambrisentan in plasma. The analyte and the internal standard (armodafinil) were extracted from plasma by acetonitrile precipitation and they were separated on a reversed-phase C(18) column with a gradient program. The MS acquisition was performed with multiple reaction monitoring mode using the respective [M + H](+) ions, m/z 379-347 for ambrisentan and m/z 274-167 for the IS. The assay exhibited a linear dynamic range of 1-2000 ng/mL for ambrisentan in plasma. Acceptable precision (<10%) and accuracy (100 ± 8%) were obtained for concentrations over the standard curve range. The method was successfully applied to quantify ambrisentan concentrations in a rodent pharmacokinetic study after a single oral administration of ambrisentan at 2.5 mg/kg to rats. Following oral administration the maximum mean concentration in plasma (C(max) ; 1197 ± 179 ng/mL) was achieved at 1.0 ± 0.9 h (T(max) ), and the area under the curve (AUC) was 6013 ± 997 ng h/mL. Therefore, development of such a simple and sensitive method in rat plasma should translate into a method for ambrisentan in human plasma for clinical trials.

Rapid Determination of Fumonisins B1 and B2 in Corn by Liquid Chromatography-Tandem Mass Spectrometry with Ultrasonic Extraction

A simple, sensitive, and reliable analytical method is developed for the rapid determination of fumonisin B(1) and fumonisin B(2) in corn by high-performance liquid chromatography-positive electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS). Fumonisin B(1) and fumonisin B(2) are extracted from corn with methanol-water (3:1, v/v) by means of ultrasonic extraction, and directly injected into an LC-MS-MS system after centrifugation. Fumonisin B(1) and fumonisin B(2) are separated on a Zorbax Eclipse XDB-C(18) column with a solution of methanol-water-formic acid as the mobile phase. The method is validated with respect to linearity, accuracy, precision, specificity, and stability. Moreover, the method was applied to real samples and demonstrated to be suitable for the determination of fumonisin B(1) and fumonisin B(2) in corn. The total time required for the analysis of one sample was ~30 min.

Multiresidue Pesticide Analysis of Agricultural Commodities Using Acetonitrile Salt-Out Extraction, Dispersive Solid-Phase Sample Clean-Up, and High-Performance Liquid Chromatography–Tandem Mass Spectrometry

A multiresidue method analyzing 209 pesticides in 24 agricultural commodities has been developed and validated using the original Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) procedure and high performance liquid chromatography-positive electrospray ionization-tandem mass spectrometry (LC-MS/MS) analysis. Using solvent-only calibration standards (SOCSs) and matrix-matched calibration standards (MMCSs), it was demonstrated that a minimal concentration of 5-10 μg/kg (part per billion, ppb) of analytes in matrix is required for the consistent identification of targeted pesticides with two MRM transitions. Method performance was validated by the precision and accuracy results obtained from fortification studies at 10, 25, 100, and 500 ppb and MMCSs. The method was demonstrated to achieve an average recovery of 100 ± 20% (n = 4) for >75% of evaluated pesticides at the low fortification level (10 ppb) and improved to >84% at the higher fortification concentrations in all 24 matrices. Matrix effects in LC-MS/MS analysis were studied by evaluating the slope ratios of calibration curves (1.0-100 ng/mL) obtained from the SOCSs and MMCSs. Principal component analysis (PCA) of LC-MS/MS and method validation data confirmed that each matrix exerts its specific effect during the sample preparation and LC-MS/MS analysis. The matrix effect is primarily dependent on the matrix type, pesticide type and concentration. Some caution is warranted when using matrix matched calibration curves for the quantitation of pesticides to alleviate concerns on matrix effects. The QuEChERS method with LC-MS/MS was used to identify and quantitate pesticides residues, with concentrations ranging from 2.5 to >1000 ppb in a variety of agricultural samples, demonstrating fitness for screening and surveillance applications.

Quantification of cinacalcet by LC–MS/MS using liquid–liquid extraction from 50 μL of plasma

A simple and economical high-performance liquid chromatography–positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of cinacalcet in plasma. Following liquid–liquid extraction, the analyte was separated using an isocratic mobile phase on a reversed-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M+H] + ions, m/ z 358–155 for cinacalcet and m/ z 310–148 for the internal standard. The assay exhibited a linear dynamic range of 0.1–200 ng/mL for cinacalcet in plasma. Acceptable precision (<10%) and accuracy (100 ± 5%) were obtained for concentrations over the standard curve range. A run time of 3.5 min for each sample made it possible to analyze more than 250 samples per day. The method was successfully applied to quantify cinacalcet concentrations in a preclinical pharmacokinetic study after a single oral administration of cinacalcet at 10 mg/kg to rats. Following oral administration the maximum mean concentration in plasma ( C max; 160 ± 56 ng/mL) was achieved at 1.0 h ( T max), area under the curve (AUC) and half-life ( t 1/2) were 949 ± 257 ng h/mL and 3.58 ± 0.4 h, respectively.

Quantification of methyllycaconitine, selective α7 nicotinic receptor antagonist, in rodent plasma and brain tissue by liquid chromatography tandem mass spectrometry – application to neuropharmacokinetics of methyllycaconitine in rats

A sensitive high-performance liquid chromatography-positive ion electrospray tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of methyllycaconitine (MLA) in rat plasma and brain tissue. Following acetonitrile protein precipitation, the analyte was separated using a gradient mobile phase on a reversed-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M + H](+) ions, m/z 683-216 for MLA and m/z 260-116 for the internal standard. The assay exhibited a linear dynamic range of 0.5-250 ng/mL for MLA in rat plasma and brain tissue. The lower limit of quantification was 0.5 ng/mL. Acceptable precision (<12%) and accuracy (100 ± 6%) were obtained for concentrations over the standard curve range. The method was successfully applied to quantify MLA concentrations in a rodent pharmacokinetic and brain penetration study.

Quantification of urapidil, α-1-adrenoreceptor antagonist, in plasma by LC-MS/MS: validation and application to pharmacokinetic studies

A sensitive high-performance liquid chromatography-positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of urapidil in plasma. Following liquid-liquid extraction, the analyte was separated using an isocratic mobile phase on a reverse-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M + H](+) ions, m/z 388 to 205 for urapidil and m/z 452 to 344 for the internal standard. The assay exhibited a linear dynamic range of 0.1-500 ng/mL for urapidil in plasma. Acceptable precision (<7%) and accuracy (100 ± 8%) were obtained for concentrations over the standard curve range. The method was successfully applied to quantify urapidil concentrations in a preclinical pharmacokinetic study after a single oral administration of urapidil at 3 mg/kg to rats. Following oral administration the maximum mean concentration in plasma (C(max); 616 ± 73 ng/mL) was achieved at 0.5 h (T(max)) and area under curve (AUC(0-24)) was 1841 ± 308 ng h/mL. The half-life (t(1/2)) and clearance (Cl) were 2.47 ± 0.4 h and 1660 ± 276 mL/h/kg, respectively. Moreover, it is plausible that the assay method in rat plasma would facilitate the adaptability of urapidil quantification in human plasma for clinical trials.

A Rapid and Selective LC‐MS/MS Method for Quantification of Quetiapine in Human Plasma and its Application to Pharmacokinetic Study on Indian Schizophrenia Patients

A rapid, robust and selective high pressure liquid chromatography–positive electrospray ionization tandem mass spectrometry method has been developed and validated for the quantification of quetiapine (QUE) in human plasma with K2EDTA using oxcarbazepine (IS) as an internal standard. Analyte and internal standard were extracted from human plasma by solid‐phase extraction using acetonitrile. The eluted samples were chromatographed on a C18 column by using a 10:75:15v/v mixture of ammonium formate buffer (5 mM, pH 4.50) and acetonitrile and methanol as an isocratic mobile phase at a flow rate of 0.4 mL/min and analyzed by mass spectrometry in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 384.3/253.2 for Quetiapine and m/z 253.1/208.1 for the internal standard. The assay exhibited a linear dynamic range of 5.01 ‐ 2501.04 ng/mL for quetiapine in human plasma. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.5 min for each sample made it possible to analyze 300 patient plasma samples per day. The validated method has been successfully used for the estimation of quetiapine in real time schizophrenia patient’s plasma samples for pharmacokinetic study.

Simultaneous determination of clenbuterol, salbutamol and ractopamine in milk by reversed-phase liquid chromatography tandem mass spectrometry with isotope dilution

A simple, sensitive and reliable analytical method was developed for the simultaneous determination of clenbuterol (CLB), salbutamol (SAL) and ractopamine (RAC) in milk by ultra high performance liquid chromatography–positive electrospray ionization tandem mass spectrometry (UHPLC–ESI-MS/MS) with isotope dilution. Samples were directly purified through HLB cartridge. Then the eluate was dried under nitrogen and residues were redissolved in mobile phase. Samples were analyzed by LC–MS/MS on an Acquity UPLC ® BEH C 18 column with gradient elution. The samples were quantified using clenbuterol-D 9, salbutamol-D 3 and ractopamine-D 6 as internal standards. The proposed method was validated according to the European Commission Decision 2002/657/EC determining specificity, decision limit (CCα), detection capability (CCβ), recovery, precision, linearity, robustness and stability. CCα values were 0.054, 0.006 and 0.008 μg/kg for CLB, SAL and RAC, respectively. CCβ values were 0.058, 0.007 and 0.009 μg/kg for CLB, SAL and RAC, respectively. The mean recoveries, repeatability (expressed as coefficient of variation, CV r), and reproducibility (CV R) varied from 95.8 to 106.2%, from 3.60 to 6.44% (CVr), and from 4.77 to 7.53% (CV R), respectively. The method is demonstrated to be suitable for the determination of clenbuterol, salbutamol and ractopamine in milk. The total time required for the analysis of one sample, including sample preparation, was about 45 min.