Development and validation of a Sensitive bioanalytical method for the quantitative estimation of Pantoprazole in human plasma samples by LC–MS/MS: Application to bioequivalence study

The present study aims at developing a simple, sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the quantification of pantoprazole sodium (PS) in human plasma using pantoprazole D3 (PSD3) as internal standard (IS). Chromatographic separation was performed on Zorbax SB-C18, 4.6 mm × 75 mm, 3.5 μm, 80 Å column with an isocratic mobile phase composed of 10 mM ammonium acetate (pH 7.10): acetonitrile (30:70, v/v), pumped at 0.6 mL/min. PS and PSD3 were detected with proton adducts at m/z 384.2 → 200.1 and 387.1 → 203.1 in multiple reaction monitoring (MRM) positive mode, respectively. Precipitation method was employed in the extraction of PS and PSD3 from the biological matrix. This method was validated over a linear concentration range of 10.00–3000.00 ng/mL with correlation coefficient (r) ≥ 0.9997. Intra- and inter-day precision of PS were found to be within the range of 1.13–1.54 and 1.76–2.86, respectively. Both analytes were stable throughout freeze/thaw cycles, bench top and postoperative stability studies. This method was successfully utilized in the analysis of blood samples following oral administration of PS (40 mg) in healthy human volunteers.     

Development,validation and comparison of LC–MS/MS and RIA methods for quantification of vertebrates-like sex-steroids in prosobranch molluscs

The role of vertebrate-like sex-steroids (testosterone, T, progesterone, P, and 17β-estradiol, E2) in molluscs is still debated, but they could represent potential biomarkers of endocrine disruption. A radioimmunoassay (RIA) and a liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) methods have been developed and compared to measure their levels in a gastropod snail Potamopyrgus antipodarum. Both methods showed a good reproducibility despite the complex matrix and the very low levels of vertebrate-like sex-steroids. Only T and P were detected using the LC–MS/MS method, while the RIA method reached lower detection limits and enabled the detection of all three steroids. Results indicated that T and P were mainly present as unconjugated forms. Both methods were compared in the analysis of snails exposed to waste water treatment plant effluents and led to the same conclusions concerning the modulation of steroids levels. Moreover, they both were in agreement concerning T measurements. On the other hand, LC–MS/MS appeared to be more suitable when analyzing P levels due to a low sensitivity of the RIA method. As E2 was not measured using the LC–MS/MS method because of a higher detection limit compared to the other steroids, the results obtained with the RIA method should be interpreted with caution. LC–MS/MS remains the gold standard for sex-steroid determinations, however a relevant and alternative method based on RIA was developed, requiring fewer organisms. RIA seems a promising method as a screening tool for experimental use, allowing comparison of sex-steroid levels in the mudsnail both in laboratory and in field experiments.     

Development and validation of a LC–MS/MS method for the determination of clebopride and its application to a pharmacokinetics study in healthy Chinese volunteers

A sensitive and specific liquid chromatography–electrospray ionization-mass spectrometry (LC–ESI-MS/MS) method has been developed and validated for the identification and quantification of clebopride in human plasma using itopride as an internal standard. The method involves a simple liquid–liquid extraction. The analytes were separated by isocratic gradient elution on a CAPCELL MG-III C₁₈ (5 μm, 150 mm × 2.1 mm i.d.) column and analyzed in multiple reaction monitoring (MRM) mode with positive electrospray ionization (ESI) interface using the respective [M+H]⁺ ions, m/z 373.9 → m/z184.0 for clebopride, m/z 359.9 → m/z71.5 for itopride. The method was validated over the concentration range of 69.530–4450.0 pg/ml for clebopride. Within- and between-batch precision (RSD%) was all within 6.83% and accuracy ranged from −8.16 to 1.88%. The LLOQ was 69.530 pg/ml. The extraction recovery was on an average 77% for clebopride. The validated method was used to study the pharmacokinetics profile of clebopride in human plasma after oral administration of clebopride.     

LC–MS/MS method development and validation for the determination of polymyxins and vancomycin in rat plasma

Simple, sensitive and robust liquid chromatography–tandem mass spectrometer (LC–MS/MS) methods were developed and validated for the determination of lipopeptide polymyxins and glycopeptide vancomycin in rat plasma. The effect of trichloroacetic acid (TCA) concentration on sample recoveries (peak area of sample recovered from plasma/peak area of sample from neat solvent solutions) was studied and an optimized concentration of 30% TCA were determined that gives the best sample recovery for the peptides from rat plasma. The effect of the TCA concentration on the chromatographic behavior of peptides was studied on a Phenomenex Jupiter C18 5 μ 300 Å 50 mm × 2 mm column using a mobile phase with a pH of 2.8. Other than protein precipitation, TCA also acted as ion pairing reagent and was only present in the samples but not in the mobile phases. The data demonstrated that by increasing the TCA concentration, the analyte retention and sensitivity were improved. The absence of TCA in mobile phase helped to reduce the ion source contamination and to achieve good reproducibility. The plasma method was linearly calibrated from 5 to 5000 ng/mL for polymyxins with precisions to be of 2.3–10.8%, and accuracies to be 91.7–107.4% for polymyxin B1, B2, E1, E2, respectively. For vancomycin the calibration is from 1 to 5000 ng/mL with precisions to be of 7.8–10.3 and accuracies to be 96.2–102.0%. The LLOQs corresponding with a coefficient of variation less than 20% were 7.5, 18.1, 7.3, 5.0 and 1.0 ng/mL for polymyxin B1, B2, E1, E2 and vancomycin, respectively.     

Development and validation of a sensitive and rapid non-aqueous LC–ESI-MS/MS method for measurement of diosgenin in the plasma of normal and hyperlipidemic rats: A comparative study

A sensitive and specific electrospray ionization liquid chromatography–tandem mass spectrometry method was developed to detect diosgenin in the plasma of normal and hyperlipidemic rats. Diosgenin was extracted with n-hexane–ethyl acetate (9:1, v/v) using sarsasapogenin as an internal standard. With multiple reaction monitoring modes, linear calibration curves were obtained in the range 10–1500 ng/mL (r ≥ 0.9979) and the limit of quantification was 10 ng/mL. Intra- and inter-assay variabilities were within 7.74%, and accuracies were between ?5.33% and 1.50%. The assay was successfully applied to study pharmacokinetics in rats after oral administration of diosgenin. Significantly different pharmacokinetics between normal and hyperlipidemic rats were observed, which would be beneficial for the clinical use of diosgenin.     

Development and validation of a high throughput and robust LC–MS/MS with electrospray ionization method for simultaneous quantitation of diltiazem and its two metabolites in human plasma: Application to a bioequivalence study

A high throughput and specific method using ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) was developed for the simultaneous determination of diltiazem and its two metabolite (N-desmethyldiltiazem and O-desacetyldiltiazem) in human plasma. A one-step liquid–liquid extraction (LLE) with methyl-t-butyl ether (MTBE) involved for the extraction of diltiazem (DLTZ), metabolites (DMeD and DAcD) and internal standard. Analytes were chromatographed on a ACQUITY UPLC? BEH C₁₈ column (100 mm × 2.1 mm, i.d., 1.7 μm) with isocratic elution at a flow rate of 0.2 mL/min using 10 mM ammonium acetate buffer–acetonitrile (25:75, v/v). The Quattro Premier XE LC–MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. Using 300 μL plasma, the method was validated over the concentration range 0.48–639.9 ng/mL for DLTZ and 0.24–320.1 for DMeD and 0.24–320.7 ng/mL for DAcD, with a lower limit of quantification of 0.48 ng/mL for DLTZ and 0.24 ng/mL for metabolites. The intra- and inter-day precision and accuracy were within 10.0%. The recovery was 77.4%, 76.0%, 74.5% and 74.1% for DLTZ, DMeD, DAcD and Ziprasidone, respectively. Total run time was 2.0 min only.     

Rapid and sensitive LC–MS/MS assay for the quantitation of 20(S)-protopanaxadiol in human plasma

This paper describes a rapid and sensitive method for the quantitation of 20(S)-protopanaxadiol (PPD) in human plasma based on high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS). The analyte and internal standard (I.S.), ginsenoside Rh₂, were extracted from plasma by liquid–liquid extraction and separated on a Zorbax extend C₁₈ analytical column using methanol–acetonitrile-10 mM ammonium acetate (47.5:47.5:5, v/v/v) as mobile phase. Detection was by tandem mass spectrometry using electrospray ionization in the positive ion mode and multiple reaction monitoring (MRM). The assay was linear over the concentration range 0.1–100.0 ng/ml with a limit of detection of 0.05 ng/ml. The method was successfully applied to a clinical pharmacokinetic study in healthy volunteers after a single oral administration of a PPD 25 mg capsule.     

Development and validation of an LC–MS/MS method for quantification of cyclic guanosine 3′,5′-monophosphate (cGMP) in clinical applications: A comparison with a EIA method

An LC–MS/MS method was developed and validated to quantify endogenous cyclic guanosine 3′,5′-monophosphate (cGMP) in human plasma. The LC–MS/MS and competitive enzyme immunoassay (EIA) assays were compared. cGMP concentrations of 20 human plasma samples were measured by both methods. For the MS-based assay, plasma samples were subjected to a simple protein precipitation procedure by acetonitrile prior to analysis by electrospray ionization LC–MS/MS. De-protonated analytes generated in negative ionization mode were monitored through multiple reaction monitoring (MRM). A stable isotope-labeled internal standard, ¹³C₁₀,¹⁵N₅-cGMP, which was biosynthesized in-house, was used in the LC–MS/MS method. The competitive EIA was validated using a commercially available cGMP fluorescence assay kit. The intra-assay accuracy and precision for MS-based assay for cGMP were 6–10.1% CV and ?3.6% to 7.3% relative error (RE), respectively, while inter-assay precision and accuracy were 5.6–8.1% CV and ?2.1% to 6.3% RE, respectively. The intra-assay accuracy and precision for EIA were 17.9–27.1% CV and ?4.9% to 24.5% RE, respectively, while inter-assay precision and accuracy were 15.1–39.5% CV and ?30.8% to 4.37% RE, respectively. Near the lower limits of detection, there was little correlation between the cGMP concentration values in human plasma generated by these two methods (R² = 0.197, P = 0.05). Overall, the MS-based assay offered better selectivity, recovery, precision and accuracy over a linear range of 0.5–20 ng/mL. The LC–MS/MS method provides an effective tool for the quantitation of cGMP to support clinical mechanistic studies of curative pharmaceuticals.     

Development of an LC–MS/MS assay to determine plasma pharmacokinetics of the radioprotectant octadecyl thiophosphate (OTP) in monkeys

Octadecenyl thiophosphate (OTP), a synthetic analogue of the lysophospholipid growth factor lysophosphatidic acid (LPA), significantly reduces mortality following a lethal dose of LD_80/30 radiation exposure in a mouse model of whole-body irradiation. To facilitate dose scaling between species, we developed a novel liquid chromatography/tandem mass spectrometry (LC–MS/MS) for the preclinical pharmacokinetic characterization of OTP in monkeys. Sample extraction was carried out using a butanol based liquid–liquid extraction method. A partially deuterated OTP analogue was used as internal standard (IS). OTP and IS were separated by reversed-phase liquid chromatography on a C-8 column using 10 mM ammonium acetate and acetonitrile. A triple quadrupole mass spectrometer operating in the negative electrospray ionization mode with multiple reaction monitoring was used to detect OTP and IS transitions of m/z 363.1 → 95.0 and 403.1 → 95.0. The method was applied to determine pharmacokinetic parameters in monkeys receiving a single oral OTP dose (3 mg/kg). OTP is readily absorbed with a relatively long half-life which supports further preclinical testing of OTP as a radioprotectant in monkeys.     

Development and validation of an HPLC–FLD method for milbemectin quantification in dog plasma

Milbemectin is a widely used veterinary antiparasitic agent. A high-performance liquid chromatography with fluorescent detection (HPLC–FLD) method is described for the determination of milbemectin in dog plasma. The derivative procedure included mixing 1-methylimizole [MI, MI-ACN (1:1, v/v), 100 μL], trifluoroacetic anhydride [TFAA, TFAA-ACN (1:2, v/v), 150 μL] with a subsequent incubation for 3 s at the room temperature to obtain a fluorescent derivative, which is reproducible in different blood samples and the derivatives proved to be stable for at least 80 h at room temperature. HPLC method was developed on C18 column with FLD detection at an excitation wavelength of 365 nm and emission wavelength of 475 nm, with the mobile phase consisting of methanol and water in the ratio of 98:2 (v/v). The assay lower limit of quantification was 1 ng/mL. The calibration curve was linear over concentration range of 1–200 ng/mL. The intra- and inter-day accuracy was >94% and precision expressed as % coefficient of variation was <5%. This method is specific, simple, accurate, precise and easily adaptable to measure milbemycin in blood of other animals.     

Development and validation of a liquid chromatography–tandem mass spectrometry method for the determination of goserelin in rabbit plasma

A rapid and sensitive liquid chromatography–electrospray ionization tandem mass spectrometry method (LC–ESI-MS/MS) was developed and validated for the determination of goserelin in rabbit plasma. Various parameters affecting plasma sample preparation, LC separation, and MS/MS detection were investigated, and optimized conditions were identified. Acidified plasma samples were applied to Oasis^® HLB solid-phase extraction (SPE) cartridges. Extracted samples were evaporated under a stream of nitrogen and then reconstituted with 100 μL mobile phase A. The separation was achieved on a Capcell-Pak C18 (2.0 mm × 150 mm, 5 μm, AQ type) column with a gradient elution of solvent A (0.05% acetic acid in deionized water/acetonitrile = 85/15; v/v) and solvent B (acetonitrile) at a flow rate of 250 μL/min. The LC–MS/MS system was equipped with an electrospray ion source operating in positive ion mode. Multiple-reaction monitoring (MRM) of the precursor–product ion transitions consisted of m/z 635.7 → m/z 607.5 for goserelin and m/z 424.0 → m/z 292.1 for cephapirin (internal standard). The proposed method was validated by assessing specificity, linearity, limit of quantification (LOQ), intra- and inter-day precision and accuracy, recovery, and stability. Linear calibration curves were obtained in the concentration range of 0.1–20 ng/mL (the correlation coefficients were above 0.99). The LOQ of the method was 0.1 ng/mL. Results obtained from the validation study of goserelin showed good accuracy and precision at concentrations of 0.1, 1, 5, 10, and 20 ng/mL. The validated method was successfully applied to a pharmacokinetic study of goserelin after a single subcutaneous injection of 3.6 mg of goserelin in healthy white rabbits.     

Development and validation of a sensitive gas chromatography–ammonia chemical ionization mass spectrometry method for the determination of tabun enantiomers in hemolysed blood and plasma of different species

The aim of this study was to develop and validate a fast, sensitive and easily applicable GC–MS assay for the chiral quantification of the highly toxic organophosphorus compound tabun (O-ethyl-N,N-dimethylphosphoramidocyanidate, GA) in hemolysed swine blood for further use in toxicokinetic and toxicodynamic studies. These requirements were fulfilled best by a GC–MS assay with positive chemical ionization with ammonia (GC–PCI-MS). Separation was carried out on a β-cyclodextrin capillary column (Supelco BetaDex^® 225) after reversed phase (C18) solid-phase extraction. The limit of detection was 1 pg/ml for each enantiomer (approximately 500 fg on column) and the limit of quantification 5 pg/ml. The GC–PCI-MS method was applied for the quantification of tabun enantiomers in spiked swine blood after hemolysis and in spiked plasma of different species including humans.     

Utilization of dried blood spots within drug discovery: modification of a standard DiLab(®) AccuSampler(®) to facilitate automatic dried blood spot sampling

The use of dried blood spots (DBS) in preclinical studies has seen an enormous increase over the past two years. Despite its positive impact on the 3Rs (reduce, replace and refine), its uptake in exploratory drug discovery has been limited due mainly to protracted method development time in bioanalysis but also the need for small volumes (<20 μL) to be sampled manually. Automatic blood sampling technology such as the DiLab(?) AccuSampler(?) is widely used in drug discovery to facilitate exploratory rodent-based pharmacokinetic and pharmacokinetic/pharmacodynamic studies with minimal animal handling. Propranolol was orally administered to a Han-Wistar rat attached to either a standard DiLab(?) AccuSampler(?) or a retrofitted unit designed to directly collect the DBS samples. In all, 50 or 20 μL blood samples were then collected via the standard or retrofitted unit, respectively, at six timepoints over a 7 h period. After drying and storage the DBS samples were analysed for propranolol via liquid chromatography-mass spectrometry. In this report we demonstrate that a standard DiLab(?) AccuSampler(?) can be easily retrofitted to facilitate automatic dried blood spot sampling and that time-concentration data generated from these samples are equivalent to that from manually spotted samples.     

Development and validation of sensitive LC-MS/MS assays for quantification of HP-β-CD in human plasma and CSF

2-Hydroxypropyl-β-cyclodextrin (HP-β-CD), a widely used excipient for drug formulation, has emerged as an investigational new drug for the treatment of Niemann-Pick type C1 (NPC1) disease, a neurodegenerative cholesterol storage disorder. Development of a sensitive quantitative LC-MS/MS assay to monitor the pharmacokinetics (PKs) of HP-β-CD required for clinical trials has been challenging owing to the dispersity of the HP-β-CD. To support a phase 1 clinical trial for ICV delivery of HP-β-CD in NPC1 patients, novel methods for quantification of HP-β-CD in human plasma and cerebrospinal fluid (CSF) using LC-MS/MS were developed and validated: a 2D-LC-in-source fragmentation-MS/MS (2D-LC-IF-MS/MS) assay and a reversed phase ultra performance LC-MS/MS (RP-UPLC-MS/MS) assay. In both assays, protein precipitation and “dilute and shoot” procedures were used to process plasma and CSF, respectively. The assays were fully validated and in close agreement, and allowed determination of PK parameters for HP-β-CD. The LC-MS/MS methods are ∼100-fold more sensitive than the current HPLC assay, and were successfully employed to analyze HP-β-CD in human plasma and CSF samples to support the phase 1 clinical trial of HP-β-CD in NPC1 patients.     

Development and validation of sensitive and selective LC–MS/MS methods for the determination of BMS-708163, a γ-secretase inhibitor,in plasma and cerebrospinal fluid using deprotonated or formate adduct ions as precursor ions

BMS-708163 is a γ-secretase inhibitor that is being developed for the treatment of Alzheimer's disease. Several LC–MS/MS methods have been developed for the determination of BMS-708163 in both plasma and cerebrospinal fluid in support of dog, rat, mouse and human studies. To support non-clinical studies, an LC–MS/MS method with a lower limit of quantitation (LLOQ) of 5 ng/mL, was developed and validated in dog, rat, and mouse plasma by using the deprotonated ion as the precursor ion. To support clinical studies, an LC–MS/MS method with LLOQ of 0.1 ng/mL, was developed and validated in human plasma by using the formate adduct as the precursor ion. Formic acid (0.01%) in water and acetonitrile was found to be the most favorable mobile phases for both deprotonated and formate adduct ions in negative electrospray ionization mode. A combination of a 3M Empore™ C18 plate for SPE and a Waters Atlantis dC18 analytical column for separation was used to achieve a highly selective solid phase extraction and chromatographic procedure from plasma without dry down and reconstitution steps. In the development of an assay for BMS-708163 in cerebral spinal fluid (CSF), significant non-specific binding of BMS-708163 was observed and resolved with pre- or post-spike of 0.2% Tween 20 into CSF samples. A dilute-and-shoot LC–MS/MS method with LLOQ of 0.1 ng/mL was developed and validated to assess BMS-708163 exposure in human CSF.     

Quantification of isradipine in human plasma using LC–MS/MS for pharmacokinetic and bioequivalence study

A highly sensitive and rapid method for the analysis of isradipine in human plasma using liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) was developed. The procedure involves a simple liquid–liquid extraction of isradipine and amlodipine (IS, internal standard) with methyl-t-butyl ether after alkaline treatment and separation by RP-HPLC. Detection was performed by positive ion electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode, monitoring the transitions m/z 372.1 → m/z 312.2 and m/z 408.8 → m/z 237.9, for quantification of isradipine and IS, respectively. The standard calibration curves showed good linearity within the range of 10 to 5000 pg/mL (r² ≥ 0.9998). The lower limit of quantitation (LLOQ) was 10 pg/mL. The retention times of isradipine (0.81 min) and IS (0.65 min) suggested the potential for high throughput of the proposed method. In addition, 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 studies of 5 mg of sustained-release isradipine in 24 healthy Korean volunteers.     

Development and validation of a LC–MS/MS method for the determination of the novel oral 1,14 substituted taxane derivatives,IDN 5738 and IDN 5839, in mouse plasma and its application to the pharmacokinetic study

Two LC-ESI–MS and CID-MS/MS methods were developed and validated for pharmacokinetic studies of the novel oral taxane derivatives IDN 5738 and IDN 5839, used for preclinical evaluation in mice. The analysis requires 100 μL of plasma sample, involves the addition of an internal standard and protein precipitation with 0.1% HCOOH in acetonitrile. The HPLC separation was obtained on Sunfire C18 column and Selected Reaction Monitoring technique was used to quantify the taxanes. The recoveries were more than 90%; the methods were linear over the validated concentrations range of 25–1500 ng/mL for IDN 5738 and 25–5000 ng/mL for IDN 5839 and had a limit of detection of 0.14 and 0.25 ng/mL, respectively. The inter-day coefficient of variation (CV%) of the calibration standards ranged between 1.3 and 7.2% for IDN 5738 and between 0.0 and 9.0% for IDN 5839 and the mean accuracy was in the range 85.3–112.0% for IDN 5738 and between 80.0 and 111.0% for IDN 5839. Moreover, analysing quality control plasma samples on three different days, the methods resulted precise and accurate showing intra- and inter-day CV within 12% for both analytes, and accuracy of 92.0–113.3% and 85.9–105.7% for IDN 5738 and IDN 5839, respectively. With these methods, we studied for the first time, the pharmacokinetics of the two taxanes showing for both, good oral bioavailability (>50%).     

Column-switching HPLC–MS/MS analysis of ropivacaine in serum,ultrafiltrate and drainage blood for validating the safety of blood reinfusion

A high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS–MS) method, using back-flush column-switching was developed for total drug concentrations of ropivacaine in serum and drainage blood in the measuring range 0.1–10 μg/mL. Samples were diluted with internal standard (²H₇-ropivacaine) and extraction buffer, centrifuged and injected directly onto a BioTrap 500 MS extraction column. Using a time programmed six-port valve switch, ropivacaine was back-flushed onto a Zorbax SB-Aq analytical column, gradient eluted and finally detected after electro spray ionisation and multiple reaction monitoring (MRM) of the transitions m/z 275 → m/z 126 and m/z 282 → m/z 133 for ropivacaine and ²H₇-ropivacaine, respectively. Accuracy (bias-%) was −1.5 to 5.8% and intermediate precision (C.V.) was 1.4–3.1%. The low sample amount required (10 μL), high specificity and short run time (6 min) makes it very suitable for determination of ropivacaine. Using the same methodology as described above and 200 μL ultrafiltrate, the free drug concentrations of ropivacaine in serum could be precisely determined with a C.V. below 3%. The method was used to investigate the safety of reinfusion of drainage blood after knee and hip arthroplasty when ropivacaine (Naropin^®) was used for local analgesia. Data for 30 patients are summarised.