A simple and sensitive LC‐MS/MS method for the determination of sotalol in rat plasma

A sensitive, rapid and robust HPLC method with tandem mass spectrometry (HPLC/MS/MS) detection has been developed and validated for the quantification of sotalol in rat plasma. Plasma samples were precipitated with acetonitrile before analysis. The chromatographic separation was performed on an Atlantis hydrophilic interaction liquid chromatography Silica column (50 × 2.1 mm, 3 µm) with a gradient mobile phase of 10 mm NH₄COOH (containing 0.2% of formic acid) as buffer A and acetonitrile as mobile phase B. Sotalol (m/z 273.2 → 255.1) and atenolol (the internal standard, IS, m/z 267.2 → 190.1) were monitored under positive ionization mode with 5500 QTRAP. Retention time of sotalol and the IS were 2.69 and 3.43 min, respectively. The linear range was 5–500 nm based on the analysis of 0.1 mL of plasma. The intrabatch precision ranged from 1.2 to 6.1%, and the inter‐batch precision was from 3.3 to 6.5%. The coefficient of variation of IS‐normalized matrix factor was 7.6%. Experiments for stability were performed and the analyte was sufficiently stable. A run time of 6 min for each injection made it possible to analyze a high throughput of plasma samples. The assay was successfully applied to the determination of sotalol in rat plasma after a micro‐dose oral administration. Copyright © 2014 John Wiley & Sons, Ltd.     

A sensitive LC‐MS/MS method for the quantification of febuxostat in human plasma and its pharmacokinetic application

An improved, simple and highly sensitive LC‐MS/MS method has been developed and validated for quantification of febuxostat with 100 μL human plasma using febuxostat‐d₇ as an internal standard (IS) according to regulatory guidelines. The analyte and IS were extracted from human plasma via liquid–liquid extraction using diethyl ether. The chromatographic separation was achieved on a Zorbax C₁₈ column using a mixture of acetonitrile and 5 mm ammonium formate (60:40, v/v) as the mobile phase at a flow rate of 0.5 mL/min. The total run time was 5.0 min and the elution of febuxostat and IS occurred at 1.0 and 1.5 min, respectively. A linear response function was established for the range of concentrations 1–6000 ng/mL (r > 0.99). The precursor to product ion transitions monitored for febuxostat and IS were m/z 317.1 → 261.1 and 324.2 → 262.1, respectively. The intra‐ and inter‐day precisions (%RSD) were within 1.29–9.19 and 2.85–7.69%, respectively. The proposed method was successfully applied to pharmacokinetic studies in humans. Copyright © 2013 John Wiley & Sons, Ltd.     

LC of sulfonamide residues in poultry muscle and eggs extracts using fluorescence pre‐column derivatization and monolithic silica column

A new, rapid, sensitive and selective HPLC method with fluorescence detection is described for the simultaneous determination of 12 sulfonamides, in the presence of putrescine as internal standard, after pre‐column derivatization with fluorescamine. The drugs were separated on a Chromolith Performance RP‐18 column (100×4.6 mm), using a gradient elution with a binary mobile phase of methanol/0.05 M acetate buffer (pH 3.4). Linearity of derivatization was obtained for concentrations from 3.0 to 300 μg/L in standard solutions. The whole procedure was evaluated and fully validated, according to the European Union Decision 2002/657/EC, for the determination of sulfonamides in turkey muscle and hen eggs following SPE. The LODs varied from 2 to 17 μg/kg in turkey and 2 to 15 μg/kg in egg samples. The average recoveries ranged between 96.9–108.6% in turkey muscle and 96.0–108.4% in egg samples, respectively.     

Simple and sensitive LC‐MS/MS‐based assay for the quantification of dimemorfan in human plasma for use in a pharmacokinetic study

Dimemorfan phosphate has been widely used for 40 years throughout the world for the treatment of coughs. This is the first report on the use of an LC‐MS/MS‐based assay for the determination of dimemorfan in human plasma using estazolam as an internal standard after one‐step protein precipitation with acetonitrile. Chromatographic separation was achieved on a Phenomenex Luna C₁₈ column (3 µm, 50 × 2.0 mm) using a fast gradient method, which involves water and methanol as the mobile phase (both containing 0.1% formic acid). Dimemorfan and estazolam were detected with proton adducts at m/z values of 255.8 → 155.1 and 295.0 → 267.0, respectively, in the selected reaction monitoring positive mode. The linear dynamic range of the assay was 0.04–5.00 ng/mL. The chromatographic run time for each plasma sample was <5 min. The method was proven to be accurate, precise, and repeatable. Finally, the developed method was successfully applied for the determination of dimemorfan in a pharmacokinetic study using healthy Chinese subjects. Copyright © 2014 John Wiley & Sons, Ltd.     

Development and validation of a sensitive assay for the quantification of imatinib using LC/LC‐MS/MS in human whole blood and cell culture

We developed and validated a semi‐automated LC/LC‐MS/MS assay for the quantification of imatinib in human whole blood and leukemia cells. After protein precipitation, samples were injected into the HPLC system and trapped onto the enrichment column (flow 5 mL/min); extracts were back‐flushed onto the analytical column. Ion transitions [M + H]⁺ of imatinib (m/z = 494.3 → 394.3) and its internal standard trazodone (372.5 → 176.3) were monitored. The range of reliable response was 0.03–75 ng/mL. The inter‐day precisions were: 8.4% (0.03 ng/mL), 7.2% (0.1 ng/mL), 6.5% (1 ng/mL), 8.2% (10 ng/mL) and 4.3% (75 ng/mL) with no interference from ion suppression. Autosampler stability was 24 hs and samples were stable over three freeze–thaw cycles. This semi‐automated method is simple with only one manual step, uses a commercially available internal standard, and has proven to be robust in larger studies. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis of benzofurazan derivatization reagents for short chain carboxylic acids in liquid chromatography/electrospray ionization‐tandem mass spectrometry (LC/ESI‐MS/MS)

Benzofurazan derivatization reagents, 4‐[2‐(N,N‐dimethylamino)ethylaminosulfonyl]‐7‐(2‐aminopentylamino)‐2,1,3‐benzoxadiazole (DAABD‐AP) and 4‐[2‐(N,N‐dimethylamino) ethylaminosulfonyl]‐7‐(2‐aminobutylamino)‐2,1,3‐benzoxadiazole (DAABD‐AB), for short‐chain carboxylic acids in liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) were synthesized. These reagents reacted with short chain carboxylic acids in the presence of the condensation reagents at 60°C for 60 min. The generated derivatives were separated on the reversed‐phase column and detected by ESI‐MS/MS with the detection limits of 0.1–0.12 pmol on column. Upon collision‐induced dissociation, a single and intense product ion at m/z 151 was observed. These results indicated that DAABD‐AP and DAABD‐AB are suitable as the derivatization reagents in LC/ESI‐MS/MS analysis. Copyright © 2008 John Wiley & Sons, Ltd.     

Determination of eflornithine enantiomers in plasma by precolumn derivatization with o‐phthalaldehyde‐N‐acetyl‐l‐cysteine and liquid chromatography with UV detection

A bioanalytical method for indirect determination of eflornithine enantiomers in 75 μL human plasma has been developed and validated. l ‐ and d ‐eflornithine were derivatized with o‐phthalaldehyde and N‐acetyl‐L‐cysteine to generate diastereomers which were separated on two serially connected Chromolith Performance columns (RP‐18e 100 × 4.6 mm i.d.) by a isocratic flow followed by a gradient flow for elution of endogenous compounds. The diastereomers were detected with UV (340 nm). The between‐day precisions for L‐ and D‐eflornithine in plasma were 8.4 and 2.3% at 3 μm , 4.0 and 5.1% at 400 μm , and 2.0 and 3.7% at 1000 μm . The lower limit of quantification was determined to be 1.5 μm , at which precision was 14.9 and 9.9% for L‐ and D‐eflornithine, respectively. Copyright © 2009 John Wiley & Sons, Ltd.     

Metabolic profile and pharmacokinetics of polyphyllin I,an anticancer candidate,in rats by UPLC‐QTOF‐MS/MS and LC‐TQ‐MS/MS

Polyphyllin I (PPI), a natural steroidal saponin originating from rihzome of Paris polyphylla , is a potential anticancer candidate. Previous pharmacokinetics study showed that the oral bioavailability of PPI was very low, which suggested that certain amount of PPI might be metabolized in vivo . However, to date, information regarding the final metabolic fates of PPI is very limited. In this study, metabolites of PPI and their pharmacokinetics in rats were investigated using UPLC‐QTOF‐MS/MS and LC‐TQ‐MS/MS. A total of seven putative metabolites, including six phase I and one phase II metabolites, were detected and identified with three exact structures by comparison with authentic standards for the first time. Oxidation, deglycosylation and glucuronidation were found to be the major metabolic processes of the compound in rats. The pharmacokinetics of prosapogenin A, trillin and diosgenin, three deglycosylation metabolites of PPI with definite anticancer effects, were further studied, which suggested that the metabolites underwent a prolonged absorption and slower elimination after intragastric administration of PPI at the dose of 500 mg/kg. This study provides valuable and new information on the metabolic fate of PPI, which will be helpful in further understanding its mechanism of action.     

Development and validation of a liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method for the quantification of tigecycline in rat brain tissues

Tigecycline (TIG), a derivative of minocycline, is the first in the novel class of glycylcyclines and is currently indicated for the treatment of complicated skin structure and intra‐abdominal infections. A selective, accurate and reversed‐phase high‐performance liquid chromatography‐tandem mass spectrometry (HPLC‐MS/MS) method was developed for the determination of TIG in rat brain tissues. Sample preparation was based on protein precipitation and solid phase extraction using Supel‐Select HLB (30 mg/1 mL) cartridges. The samples were separated on a YMC Triart C₁₈ column (150 mm x 3.0 mm. 3.0 µm) using gradient elution. Positive electrospray ionization (ESI+) was used for the detection mechanism with the multiple reaction monitoring (MRM) mode. The method was validated over the concentration range of 150–1200 ng/mL for rat brain tissue. The precision and accuracy for all brain analyses were within the acceptable limit. The mean extraction recovery in rat brain was 83.6%. This validated method was successfully applied to a pharmacokinetic study in female Sprague Dawley rats, which were given a dose of 25 mg/kg TIG intraperitoneally at various time‐points. Copyright © 2015 John Wiley & Sons, Ltd.     

Rapid quantification of levosulpiride in human plasma using RP‐HPLC‐MS/MS for pharmacokinetic and bioequivalence study

A rapid and validated method for analysis of levosulpiride in human plasma using liquid chromatography coupled to tandem mass spectrometry was developed. Levosulpiride and tiapride (IS, internal standard) were extracted from alkalized plasma samples with ethylacetate and separation by RP‐HPLC. Detection was performed by positive ion electrospray ionization in multiple‐reaction monitoring mode, monitoring the transitions m/z 342.1 → m/z 112.2 and m/z 329.1 → m/z 213.2, for quantification of levosulpiride and IS, respectively. The standard calibration curves showed good linearity within the range of 2–200 ng/mL (r² ≥ 0.9990). The lower limit of quantitation was 2 ng/mL. The retention times of levosulpiride (0.63 min) and IS (0.66 min) presented a significant time saving benefit of the proposed method. No significant metabolic compounds were found to interfere with the analysis. This method offered good precision and accuracy and was successfully applied for the pharmacokinetic and bioequivalence study of a 25 mg of levosulpiride tablet in 24 healthy Korean volunteers. Copyright © 2009 John Wiley & Sons, Ltd.     

A quantitative analysis of the polyamine in lung cancer patient fingernails by LC‐ESI‐MS/MS

A quantitative analysis of polyamines in lung cancer patient fingernails by the combination of 4‐(N,N‐dimethylaminosulfonyl)‐7‐fluoro‐2,1,3‐benzoxadiazole derivatives and liquid chromatography–electrospray ionization tandem mass spectrometry is described. The reaction of the reagent with eight kinds of polyamines, that is, N¹‐acetylputrescine (N¹‐actPUT), N⁸‐acetylspermidine, N¹‐acetylspermine, 1,3‐diaminopropane, putrescine (PUT), cadaverine, spermidine and spermine (SPM) effectively occurs at 60 °C for 30 min. The detection limits (signal‐to‐noise ratio 5) were 5–100 fmol. A good linearity was achieved from the calibration curves, which was obtained by plotting the peak area ratios of the analytes relative to the internal standard (IS), that is, 1,6‐diaminohexane, vs the injected amounts of polyamines (r² > 0.996), and the intra‐day and inter‐day assay precisions were <9.84%. Furthermore, the recoveries (%) of the polyamines spiked in the human fingernails were 89.14–110.64. The present method was applied to human fingernail samples from 17 lung cancer patients and 39 healthy volunteers. The polyamine concentration was different based on the gender, that is, the N¹‐actPUT and PUT contents were 3.10 times and 2.56 times higher in healthy men than in women, respectively. Additionally, in the lung cancer patient group, as compared with the healthy volunteers, the concentrations of SPM had a statistically significant (p < 0.05) correlation. Therefore, because the proposed method provides a good mass accuracy and the trace detection of the polyamines in human fingernails, this analytical technique could be a noninvasive technique to assist in the diagnosis and assessment of disease activity in lung cancer patients. Copyright © 2013 John Wiley & Sons, Ltd.     

A validated LC‐MS/MS assay for simultaneous quantification of methotrexate and tofacitinib in rat plasma: application to a pharmacokinetic study

A highly sensitive, specific and rapid LC‐ESI‐MS/MS method has been developed and validated for simultaneous quantification of methotrexate (MTX) and tofacitinib (TFB) in rat plasma (50 μL) using phenacetin as an internal standard (IS), as per the US Food and Drug Administration guidelines. After a solid‐phase extraction procedure, the separation of the analytes and IS was performed on a Chromolith RP_18e column using an isocratic mobile phase of 5 m m ammonium acetate (pH 5.0) and acetonitrile at a ratio of 25:75 (v/v) using flow‐gradient with a total run time of 3.5 min. The detection was performed in multiple reaction monitoring mode, using the transitions of m/z 455.2 → 308.3, m/z 313.2 → 149.2 and m/z 180.3 → 110.2 for MTX, TFB and IS, respectively. The calibration curves were linear over the range of 0.49–91.0 and 0.40–74.4 ng/mL for MTX and TFB, respectively. The intra‐ and interday accuracy and precision values for MTX and TFB were <15% at low quality control (QC), medium QC and high QC and <20% at lower limit of quantification. The validated assay was applied to derive the pharmacokinetic parameters for MTX and TFB post‐dosing of MTX and TFB orally and intravenously to rats. Copyright © 2014 John Wiley & Sons, Ltd.     

Challenges in the identification process of phenidate analogues in LC‐ESI‐MS/MS analysis: Information enhancement by derivatization with isobutyl chloroformate

A new analytical technique for the structural elucidation of four representative phenidate analogues possessing a secondary amine residue, which leads to a major/single amine‐representative fragment/product ion at m/z 84 both in their GC‐EI‐MS and LC‐ESI‐MS/MS spectra, making their identification ambiguous, was developed. The method is based on “in vial” chemical derivatization with isobutyl chloroformate in both aqueous and organic solutions, followed by liquid chromatography‐electrospray ionization mass spectrometry (LC‐ESI‐MS/MS). The resulting carbamate derivatives promote rich fragmentation patterns with full coverage of all substructures of the molecule, enabling detailed structural elucidation and unambiguous identification of the original compounds at low ng/mL levels.     

A highly sensitive and efficient UPLC‐MS/MS assay for rapid analysis of tedizolid (a novel oxazolidinone antibiotic) in plasma sample

Tedizolid (TDZ) is a novel oxazolidinone class antibiotic, indicated for the treatment of acute bacterial skin and skin structure infections in adults. In this study a highly sensitive UPLC‐MS/MS assay was developed and validated for the determination of TDZ in rat plasma using rivaroxaban as an internal standard (IS). Both TDZ and IS were separated on an Acquity UPLC BEH? C₁₈ column using an isocratic mobile phase comprising of acetonitrile–20 mm ammonium acetate (85:15, v/v), eluted at 0.3 mL/min flow rate. The plasma sample was processed by liquid liquid extraction technique using ethyl acetate as an extracting agent. The analyte and IS were detected in positive mode using electrospray ionization source. The precursor to product ion transitions at m/z 371.09 > 343.10 for TDZ and m/z 435.97 > 144.94 for IS were used for the quantification in multiple reaction monitoring mode. The calibration curve was linear in the concentration range of 0.74–1500 ng/mL and the lower limit of quantification was 0.74 ng/mL only. The developed assay was validated following standard guidelines for bioanalytical method validation (US Food and Drug Administration) and all the validation results were within the acceptable limits. The developed assay was successfully applied into a pharmacokinetic study in rats. Copyright © 2016 John Wiley & Sons, Ltd.     

Quantification of peptides using N‐terminal isotope coding and C‐terminal derivatization for sensitive analysis by micro liquid chromatography‐tandem mass spectrometry

Stable isotope‐coding coupled with mass spectrometry is a popular method for quantitative proteomics and peptide quantification. However, the efficiency of the derivatization reaction at a particular functional group, especially in complex structures, can affect accuracy. Here, we present a dual functional‐group derivatization of bioactive peptides followed by micro liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). By separating the sensitivity‐enhancement and isotope‐coding derivatization reactions, suitable chemistries can be chosen. The peptide amino groups were reductively alkylated with acetaldehyde or acetaldehyde‐d₄ to afford N‐alkylated products with different masses. This process is simple, quick and high‐yield, and accurate comparative analysis can be achieved for the mass‐differentiated peptides. Then, the carboxyl groups were derivatized with 1‐(2‐pyrimidinyl)piperazine to increase MS/MS sensitivity. Angiotensins I–IV, bradykinin and neurotensin were analyzed after online solid phase extraction by micro LC‐MS/MS. In all instances, a greater than 17‐fold increase in sensitivity was achieved, compared with the analyses of the underivatized peptides. Furthermore, the values obtained from the present method were in agreement with the result from isotope dilution quantification using isotopically labeled angiotensin I [Asp‐Arg‐(Val‐d₈)‐Tyr‐Ile‐His‐Pro‐(Phe‐d₈)‐His‐Leu]. Copyright © 2016 John Wiley & Sons, Ltd.     

Development and validation of a rapid and sensitive UPLC–MS/MS assay for the quantification of tofacitinib in human plasma

A highly sensitive, selective and rapid ultra‐performance liquid chromatography–tandem mass spectrometry method has been developed for the quantification of a Janus kinase (JAK) inhibitor, tofacitinib (TOF). The assay employed liquid–liquid extraction with methyl‐tert butyl ether to extract tofacitinib and tofacitinib‐13C3 15 N (as internal standard) from human plasma. The samples were analyzed on a UPLC BEH C₁₈ (50 × 2.1 mm, 1.7 μm) column using acetonitrile and 10.0 mm ammonium acetate, pH 4.5 (75:25, v/v) as the mobile phase within 1.4 min. The precursor/product ion transitions were monitored at m/z 313.3/149.2 and 317.4/149.2 for tofacitinib and tofacitinib‐13C3 15 N, respectively, in the positive electrospray ionization mode. The calibration curves were linear (r² ≥ 0.9978) across the concentration range of 0.05–100 ng/mL. The mean extraction recovery of tofacitinib across quality controls was 98.6%. The intra‐ and inter‐batch precision (CV) and accuracy ranged from 2.1–5.1 and 96.2–103.1%, respectively. All validation results complied well with the current guidelines. The method is amenable to high sample throughput and was applied to determine TOF plasma concentration in a pharmacokinetic study with 12 healthy Indian subjects after oral administration of 5 mg tablets.     

Development and validation of UPLC‐MS/MS method for simultaneous determination of gestodene and ethinyl estradiol in rat plasma

A selective and sensitive ultra‐performance liquid chromatography method with tandem mass spectrometric detection for simultaneous determination of gestodene (GES) and ethinyl estradiol (EE) in rat plasma was developed and validated. GES, EE and the internal standard, norgestrel, were extracted with ethyl acetate, derivatized (EE only) with dansyl chloride and then back‐extracted into diethyl ether‐hexane (2:1, v/v). The separation was performed on an ACQUITY UPLC? BEH C₁₈ column with gradient elution using mobile phase consisting of acetonitrile and water (both containing 0.1% formic acid). The detection was carried out by means of electrospray ionization (ESI) mass spectrometry in positive ion mode with multiple‐reaction monitoring. Calibration curves of GES and EE were linear (r² ≥ 0.99) over the concentration ranges 1.59–159 and 0.196–78.4 ng/mL, respectively. The intra‐ and inter‐day precisions were not more than 6.9 and 12.9% for GES and 10.6 and 9.0% for EE, and the accuracies were ?2.5–8.0% for GES, and ?7.2–0.19% for EE, respectively. The method herein described was superior to previous methods and was applicable to the pharmacokinetic study of GES and EE in rats. Copyright © 2009 John Wiley & Sons, Ltd.     

A rapid and sensitive LC‐MS/MS method for the determination of osthole in rat plasma: application to pharmacokinetic study

Osthole, a major component isolated from the fruit of Cnidium monnieri (L.) Cusson, has been widely used in traditional Chinese medicine. We developed and validated a rapid and sensitive LC‐MS/MS method for the quantification of osthole in rat plasma. Sample preparation involved simple liquid–liquid extraction by ethyl acetate after addition of imperatorin as internal standard (IS). The analyte was separated using a C₁₈ column with the mobile phase of methanol–0.1% formic acid (80:20, v/v) at a flow rate of 0.4 mL/min. The elutes were detected under positive electrospray ionization in multiple reaction monitoring mode. The method was sensitive with 0.5 ng/mL as the lower limit of detection. Good linearity was obtained over the range of 1.0–500.0 ng/mL. The intra and inter‐batch accuracy for osthole in rat plasma samples ranged from 99.5 to 108.1% and the variation was <8.9%. The stability, extraction efficiency and matrix effect were also acceptable. This method was successfully applied to the pharmacokinetic study of osthole in rat after intravenous and oral administration. Copyright © 2013 John Wiley & Sons, Ltd.     

A simple LC‐MS/MS method for quantitative analysis of underivatized neurotransmitters in rats urine: assay development,validation and application in the CUMS rat model

Many amino acid neurotransmitters in urine are associated with chronic stress as well as major depressive disorders. To better understand depression, an analytical LC‐MS/MS method for the simultaneous determination of 11 underivatized neurotransmitters (4‐aminohippurate, 5‐HIAA, glutamate, glutamine, hippurate, pimelate, proline, tryptophan, tyramine, tyrosine and valine) in a single analytical run was developed. The advantage of this method is the simple preparation in that there is no need to deconjugate the urine samples. The quantification range was 25–12,800 ng mL^?1 with >85.8% recovery for all analytes. The nocturnal urine concentrations of the 11 neurotransmitters in chronic unpredictable mild stress (CUMS) model rats and control group (n = 12) were analyzed. A series of significant changes in urinary excretion of neurotransmitters could be detected: the urinary glutamate, glutamine, hippurate and tyramine concentrations were significantly lower in the CUMS group. In addition, the urinary concentrations of tryptophan as well as tyrosine were significantly higher in chronically stressed rats. This method allows the assessment of the neurotransmitters associated with CUMS in rat urine in a single analytical run, making it suitable for implementation as a routine technique in depression research. Copyright © 2015 John Wiley & Sons, Ltd.