Journal of Chromatography B (v.1023-1024, #C)

Lithocholic acid (LCA), a hepatotoxic and carcinogenic bile acid, is metabolized to 3-ketocholanoic acid (3-KCA) by cytochrome P450 3A (CYP3A). In the present study, the objectives were to develop and validate an ultra-high performance liquid chromatography-tandem mass spectrometric (UPLC–MS/MS) method to quantify 3-KCA and apply it to the human liver microsomal CYP3A-dependent LCA 3-oxidation assay. Chromatographic separation was achieved on a Waters ACQUITY™ UPLC C18 column (50 × 2.1 mm, 1.7 μm) with a gradient system consisting of 0.1% v/v formic acid in water (solvent A) and 0.1% v/v formic acid in acetonitrile (solvent B). The retention time was 3.73 min for 3-KCA and 2.73 min for cortisol (internal standard). Positive electrospray ionization with multiple reaction monitoring (MRM) mode was used to quantify 3-KCA (m/z 375.4 → 135.2) and cortisol (m/z 363.5 → 121.0). The limit of detection of 3-KCA was 10 μM, the lower limit of quantification was 33.3 μM, and the calibration curve was linear from 0.05–10 μM with r2  > 0.99. Intra-day and inter-day accuracy and precision were <13.7%. The quality control samples were stable when assessed after 4 h at room temperature, 24 h at 4 °C, 14 days at −20 °C, and three freeze-thaw cycles. The liver microsomal matrix did not affect 3-KCA quantification. The amount of KCA formed in the human liver microsomal LCA 3-oxidation assay was linear with respect to the amount of microsomal protein (up to 40 μg) and incubation time (5–30 min). Enzyme kinetics experiment indicated that LCA 3-oxidation followed the Michaelis-Menten model with an apparent K m of 26 ± 7 μM and V max of 303 ± 50 pmol/min/mg protein. This novel UPLC–MS/MS method for quantifying 3-KCA offers a specific, sensitive, and fast approach to determine liver microsomal LCA 3-oxidation.
Keywords: Human liver microsomes; CYP3A; Lithocholic acid; 3-Ketocholanoic acid; 3-Ketolithocholic acid; Ultra-high performance liquid chromatography–tandem mass spectrometry;

We previously reported the nano-surface and molecular-orientation limited (nSMOL) proteolysis, which is a novel method for selective quantitation of monoclonal antibody Fab. The nSMOL strategy is a Fab-selective limited proteolysis which utilizes the size difference between the protease nanoparticle (200 nm) and the antibody resin pore (100 nm). Here, we applied this method to a fully validated LCMS analysis of Nivolumab in human plasma. The immunoglobulin fraction was collected using Protein A resin, which was then followed by nSMOL reaction using the FG nanoparticle surface-immobilized trypsin under a nondenaturing physiological condition at 50 °C for 7 h. After removal of resin and nanoparticles by filter centrifugation, signature peptides were separated using the ODS column liquid chromatography. The signature peptide ASGITFSNSGMHWVR from Nivolumab complementarity-determining region (CDR) and the P14R internal standard were simultaneously quantified by multiple-reaction monitoring (MRM) LCMS, with parent m/z 550.8 > fragment m/z 661.5 (y11 2+). The lower limit of quantification (LLOQ) of Nivolumab using the nSMOL method was 0.977 μg/ml, with a linear dynamic range of from 0.977 to 250 μg/ml. The intra- and inter-assay precision of LLOQ, low quality control (LQC), middle quality control (MQC), and high quality control (HQC) were 7.56–17.9% and 15.6%, 6.99–9.25% and 7.51%, 2.51–8.85% and 8.01%, and 4.78–7.33% and 6.75%, respectively. Our study demonstrates that the nSMOL bioanalysis can be utilized as a reliable method for clinical pharmacokinetic studies of Nivolumab and other antibody drugs.
Keywords: Nivolumab; Immune checkpoint inhibitor; Monoclonal antibody; nSMOL; Bioanalysis; LCMS;

Simultaneous determination of 14 antiviral drugs and relevant metabolites in chicken muscle by UPLC–MS/MS after QuEChERS preparation by Pengqian Mu; Nana Xu; Tingting Chai; Qi Jia; Zhiqiang Yin; Shuming Yang; Yongzhong Qian; Jing Qiu (17-23).
A fast method for the simultaneous determination of 14 antiviral drugs and relevant metabolites in chicken muscle by ultra-high liquid chromatography tandem mass spectrometry (UPLC–MS/MS) was developed. The analytes included anti-influenza drugs (amantadine, rimantadine, oseltamivir, oseltamivir carboxylate, memantine, arbidol, and moroxydine), anti-herpes drugs (acyclovir, ganciclovir, famciclovir, penciclovir, ribavirin and its main metabolite TCONH2), and an immunomodulator (imiquimod). The samples were pretreated using a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method. The determination of the target compounds was conducted in less than 11.0 min, and specificity was ensured by the use of multiple reaction monitoring (MRM) acquisition mode. Good linearities (R 2  > 0.9928) were obtained in the range of 0.1–100 μg/L, and the recovery rates were 56.2–113.4% with RSDs of 1.7–10.3% for intra-day precision and 2.4–8.8% for inter-day precision. The LODs and LOQs of all analytes were in the ranges of 0.02–1.0 and 0.05–2.5 μg/kg, respectively. An analysis of real samples suggested that this method is simple, sensitive, reliable and practical for the detection of antiviral drugs in animal tissue.
Keywords: Antiviral drugs; Metabolites; UPLC–MS/MS; QuEChERS method; Chicken muscle;

Liquid chromatography–tandem mass spectrometric assay for ponatinib and N-desmethyl ponatinib in mouse plasma by Rolf W. Sparidans; Anita Kort; Alfred H. Schinkel; Jan H.M. Schellens; Jos H. Beijnen (24-29).
Ponatinib is a multi-targeted third generation BCR−ABL1 tyrosine-kinase inhibitor approved for specific types of leukemia. A bioanalytical assay for this drug and its N-desmethyl metabolite in mouse plasma was developed and validated using liquid chromatography-tandem mass spectrometric (LC−MS/MS) with liquid-liquid extraction as sample pre-treatment procedure.After extraction with tert-butyl methyl ether of both analytes with their isotopically labeled internal standards and evaporation and reconstitution of the extract, compounds were separated by reversed-phase liquid chromatography under alkaline conditions. After electrospray ionization, both compounds were quantified in the selected reaction monitoring mode of a triple quadrupole mass spectrometer.The linear assay was validated in the ranges 5–5000 ng/ml for ponatinib and 1–1000 ng/ml for N-desmethyl ponatinib. Within-run (n  = 18) and between-run (3 runs; n  = 18) precisions were 10% and 12% at the lower limit of quantification for the metabolite, all other precisions were ≤8% for the metabolite and ≤6% for ponatinib. Accuracies were between 92 and 108% for both compounds in the whole calibration range. The drug was sufficiently stable under most relevant analytical conditions, only ponatinib showed more than 15% hydrolytic degradation after storage for 6 h and longer at ambient temperature in mouse plasma. Finally, the assay was successfully applied to determine plasma drug levels and study pharmacokinetics after oral administration of ponatinib to female FVB mice.
Keywords: Ponatinib; N-desmethyl ponatinib; LC−MS/MS; Mouse plasma; Liquid-liquid extraction;

Determination of dexmedetomidine in children’s plasma by ultra-performance liquid chromatography tandem mass spectrometry and application to pharmacokinetic study by Hua-Cheng Liu; Wei Sun; Cheng-Yu Wang; Wei-Yang Ying; Li-Dan Zheng; Rui-Feng Zeng; Zhe Wang; Ren-Shan Ge (30-35).
A rapid, sensitive, and selective ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) was developed and validated for the determination and pharmacokinetic investigation of dexmedetomidine in children’s plasma. Sample preparation was accomplished through a simple one-step deproteinization procedure with 0.2 mL of acetonitrile to a 0.1 mL plasma sample. Plasma samples were separated by UPLC on an Acquity UPLC BEH C18 column using a mobile phase consisting of acetonitrile-0.1% formic acid in water with gradient elution. The total run time was 3.1 min and the elution of dexmedetomidine was at 1.24 min. The detection was performed on a triple quadrupole tandem mass spectrometer in the multiple reaction-monitoring mode using the respective transitions m/z 201.3 → 95.1 for dexmedetomidine and m/z 204.2 → 98.0 for the internal standard, respectively. The calibration curve was linear over the range of 0.05–10 ng/mL with a lower limit of quantitation of 0.05 ng/mL. Mean recovery rate of dexmedetomidine in plasma was in the range of 86.7–89.1%. Intra-day and inter-day precision were both <11.6%. This method was successfully applied in pharmacokinetic study after commencement of 1.0 μg/kg dexmedetomidine infusion in children.
Keywords: Dexmedetomidine; UPLC–MS/MS; Children’s plasma; Pharmacokinetics;

A robust LC–MS/MS method for the determination of pidotimod in different biological matrixes and its application to in vivo and in vitro pharmacokinetic studies by Guangji Wang; Qian Wang; Tai Rao; Boyu Shen; Dian Kang; Yuhao Shao; Jingcheng Xiao; Huimin Chen; Yan Liang (36-43).
Pidotimod, (R)-3-[(S)-(5-oxo-2-pyrrolidinyl) carbonyl]-thiazolidine-4-carboxylic acid, was frequently used to treat children with recurrent respiratory infections. Preclinical pharmacokinetics of pidotimod was still rarely reported to date. Herein, a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated to determine pidotimod in rat plasma, tissue homogenate and Caco-2 cells. In this process, phenacetin was chosen as the internal standard due to its similarity in chromatographic and mass spectrographic characteristics with pidotimod. The plasma calibration curves were established within the concentration range of 0.01–10.00 μg/mL, and similar linear curves were built using tissue homogenate and Caco-2 cells. The calibration curves for all biological samples showed good linearity (r > 0.99) over the concentration ranges tested. The intra- and inter-day precision (RSD, %) values were below 15% and accuracy (RE, %) was ranged from −15% to 15% at all quality control levels. For plasma, tissue homogenate and Caco-2 cells, no obvious matrix effect was found, and the average recoveries were all above 75%. Thus, the method demonstrated excellent accuracy, precision and robustness for high throughput applications, and was then successfully applied to the studies of absorption in rat plasma, distribution in rat tissues and intracellular uptake characteristics in Caco-2 cells for pidotimod.
Keywords: Pidotimod; LC–MS/MS; Preclinical pharmacokinetics; Caco-2 cell;

Pesticide residues exceeding standard in green tea is a widespread problem of the world’s attention, containing organochlorine pesticides (OCPs), organophosphorus pesticides (OPPs), and pyrethroids. In this research, three dimensionally honeycomb Mg-Al layered double oxide (TDH-Mg-Al-LDO) combined with graphitized carbon black (GCB), packed as a column, was used as a novel solid phase extraction sorbent, applying in simultaneous determination of 15 pesticide residues in green tea coupled with GC–MS. Compared with different type of SPE column, it showed that TDH-Mg-Al-LDO exhibited great advantages in the extraction of 15 pesticide residues from green tea, which was seldom reported before. Different experiment conditions, such as combination order of Mg-Al-LDO and GCB, dosage of sorbents, type and volume of eluting solvent were thoroughly studied and optimized. The limits of detection (LODs) of 15 pesticides ranged from 0.9 to 24.2 ng/g and the limits of quantifications (LOQs) were ranging from 3.0 to 80.0 ng/g. The recoveries using this method at three spiked concentration levels (10, 100 and 500 ng/g for Fenthion, P,P′-DDE, O,P′-DDT, P,P′-DDD and Bifenthrin, 100, 500 and 2000 ng/g for the others) range from 71.1 to 119.0%. The relative standard deviation (RSD) was from 0.1 to 7.6% in all case. The result indicated that the proposed analytical method had been successfully applied for the simultaneous determination of 15 pesticide residues in commercial green tea.
Keywords: TDH-Mg-Al-LDO; Pesticide residues; Solid phase extraction; Green tea; GC–MS;

Lovastatin (LOV) is an antihyperlipidemic agent which exhibits low bioavailability due to its poor solubility. Therefore, a nanosuspension (NS) was developed as an efficient strategy to improve its oral bioavailability. To evaluate the pharmacokinetics of LOV-NS, a novel, sensitive, and rapid UPLC–MS/MS method was developed and validated for the simultaneous determination of LOV and its metabolite lovastatin acid (LOVA) in rat plasma. Simvastatin (IS) was chosen as the internal standard, and a liquid-liquid extraction method was used to isolate LOV and LOVA from biological matrices. The analytes were analyzed on an Acquity UPLC BEH C18 column, and a gradient program was applied at a flow rate of 0.2 mL/min. Then, a tandem quadrupole mass spectrometer coupled with a positive/negative ion-switching electrospray ionization interface was employed to detect the analytes. Quantitation of the analytes was performed in the multiple reaction monitoring mode to monitor the transitions of m/z 427.1 → 325.0 for LOV and m/z 441.1 → 325.0 for IS in the positive ion mode and m/z 421.0 → 101.0 for LOVA in the negative ion mode, respectively. The method was validated over the concentration range 0.25–500 ng/mL (r2  ≥ 0.99) for both LOV and LOVA. The intra-day and inter-day precision (RSD%) of LOV and LOVA were less than 12.87% and the accuracy (RE%) was less than 5.22%. The average extraction recoveries were 90.1% and 91.9% for LOV and LOVA, and the matrix effects were found to be between 85% and 115%. The stability study showed that both analytes were stable during the experiment. Finally, this method has been successfully applied to a pharmacokinetic study in rats following a single oral dose of 10 mg/kg LOV-NS.
Keywords: Simultaneous determination; Lovastatin; Lovastatin acid; UPLC–MS/MS; Rat plasma; Pharmacokinetics;

Source discrimination of drug residues in wastewater: The case of salbutamol by Andrea Re Depaolini; Elena Fattore; Francesca Cappelli; Raffaele Pellegrino; Sara Castiglioni; Ettore Zuccato; Roberto Fanelli; Enrico Davoli (62-67).
Analytical methods used for pharmaceuticals and drugs of abuse in sewage play a fundamental role in wastewater-based epidemiology (WBE) studies. Here quantitative analysis of drug metabolites in raw wastewaters is used to determine consumption from general population. Its great advantage in public health studies is that it gives objective, real-time data about community use of chemicals, highlighting the relationship between environmental and human health. Within a WBE study on salbutamol use in a large population, we developed a procedure to distinguish human metabolic excretion from external source of contamination, possibly industrial, in wastewaters. Salbutamol is mainly excreted as the sulphate metabolite, which is rapidly hydrolyzed to the parent compound in the environment, so this is currently not detected. When a molecule is either excreted un-metabolized or its metabolites are unstable in the environment, studies can be completed by monitoring the parent compound. In this case it is mandatory to assess whether the drug in wastewater is present because of population use or because of a specific source of contamination, such as industrial manufacturing waste. Because commercial salbutamol mainly occurs as a racemic mixture and is stereoselective in the human metabolism, the enantiomeric relative fraction (EFrel) in wastewater samples should reflect excretion, being unbalanced towards one of two enantiomers, if the drug is of metabolic origin. The procedure described involves chiral analysis of the salbutamol enantiomers by liquid chromatography-tandem mass spectrometry (LC–MS-MS) and calculation of EFrel, to detect samples where external contamination occurs. Samples were collected daily between October and December 2013 from the Milano Nosedo wastewater treatment plant. Carbamazepine and atenolol were measured in the sewage collector, as “control” drugs. Salbutamol EFrel was highly consistent in all samples during this three-month period, but a limited number of samples had unexpectedly high concentrations where the EFrel was close to that observed of the un-metabolized, commercially available drug, supporting the idea of an external source of contamination, besides human metabolic excretion. Results showed that, when present, non-metabolic daily loads could be evaluated indicating an average of 4.12 g/day of salbutamol extra load due to non-metabolic sources. The stereoselectivity in metabolism and enantiomeric ratio analysis appears to be a useful approach in WBE studies to identify different sources of drugs in the environment, when no metabolic products are present at useful analytical levels.
Keywords: Chiral analysis; Source discrimination; Salbutamol; Wastewater-based epidemiology;

A sensitive and accurate stable isotope dilution UHPLC–MS/MS method was developed and validated for the detection and quantification of ProdG adducts in human urine, a surrogate for the ProdG adducts in genomic DNA of human. A specific solid phase extraction (SPE) approach was established for selective enrichment of urinary ProdG adducts and elimination of urinary matrix facilitating the coupled MS/MS detection. The recovery of the method is estimated about 84.8–107.2%, and the precision are about 0.8–3.6% for intraday and 2.8–10.0% for interday. Due to that the matrix effect is efficiently eliminated by SPE pretreatment, the limits of detection (LODs, S/N = 3) and quantification (LOQs, S/N = 10) are decreased to 100 and 300 amol for urinary ProdG adducts, respectively. By coupling the developed SPE pretreatment with the UHPLC–MS/MS analysis, ProdG adducts were accurately quantified in healthy human urine.
Keywords: 1,N 2-propano-2′-deoxyguanosine adducts; Human urine; SPE; UHPLC–MS/MS;

The term precision of a value describes the total number of significant digits that are used to express that value. False precision is presented in data, where the sampling and chosen data file format coding return values with extra non-valid digits. The proper precision in value thus depends on so-called magnitude in digital data coding. The presence of the false precision is file format dependent. The amount of dataset points could be decreased by removing false precision (where it is present) to almost 10% of the raw measurement without loss of information.
Keywords: Mass spectrometry; Chromatography; Precision; File format; Occurency; Cumulative spectrum;