Journal of Chromatography B (v.859, #2)

Automated detection of covalent adducts to human serum albumin by immunoaffinity chromatography, on-line solution phase digestion and liquid chromatography–mass spectrometry by Johannes S. Hoos; Micaela C. Damsten; Jon S.B. de Vlieger; Jan N.M. Commandeur; Nico P.E. Vermeulen; Wilfried M.A. Niessen; Henk Lingeman; Hubertus Irth (147-156).
A generic method for the detection of covalent adducts to the cysteine-34 residue of human serum albumin (HSA) has been developed, based on an on-line combination of immunoaffinity chromatography for selective sample pre-treatment, solution phase digestion, liquid chromatography and tandem mass spectrometry. Selective anti-HSA antibodies immobilized on agarose were used for sample pre-concentration and purification of albumin from the chemically produced alkylated HSA. After elution, HSA and HSA adducts are mixed with pronase and directed to a reaction capillary kept at a digestion temperature of 70 °C. The digestion products were trapped on-line on a C18 SPE cartridge. The peptides were separated on a reversed-phase column using a gradient of organic modifier and subsequently detected using tandem mass spectrometry. Modified albumin samples consisted of synthetically alkylated HSA by the reactive metabolite of acetaminophen, N-acetyl-p-benzoquinoneimine (NAPQI), and using the alkylating agent 1-chloro-2,4-dinitrobenzene (CDNB) as reference. The resulting mixture of alkylated versus non-modified albumin has been applied to the on-line system, and alkylation of HSA is revealed by the detection of the modified marker tetra-peptide glutamine–cysteine–proline–phenylalanine (QCPF) adducts NAPQI-QCPF and CDNB-QCPF. Detection of alkylated species was enabled by the use of data comparison algorithms to distinguish between unmodified and modified HSA samples. The in-solution digestion proved to be a useful tool for enabling fast (less than 2 min) and reproducible on-line digestion of HSA. A detection limit of 1.5 μmol/L of modified HSA could be obtained by applying 10 μL of NAPQI-HSA sample.
Keywords: Affinity; On-line; Pronase; Protein adduct formation; Digestion; Protein; Albumin; LC–MS; NAPQI;

Simultaneous determination of five main active bufadienolides of Chan Su in rat plasma by liquid chromatography tandem mass spectrometry by Wen Xu; Heng Luo; Yaping Zhang; Lei Shan; Haiyun Li; Ming Yang; Runhui Liu; Weidong Zhang (157-163).
To study the pharmacokinetics of Chan Su, a sensitive and selective method was developed and validated for the determination of five main bufadienolides (cinobufagin, resibufogenin, bufalin, bufotalin and arenobufagin) in rat plasma. The analytes were extracted by liquid–liquid extraction with ethyl acetate after internal standard (IS, caudatin) spiked. The separation was performed by a ZORBAX SB-C18 column (3.5 μm, 2.1 mm × 100 mm) and a C18 guard column (5 μm, 4.0 mm × 2.0 mm) with an isocratic mobile phase consisted of acetonitrile–water–formic acid (50:50:0.05, v/v/v) at a flow rate of 0.3 mL/min. The Agilent G6410A triple quadrupole LC/MS system was operated under the multiple reaction monitoring mode (MRM) using the electrospray ionization technique in positive mode. The nominal retention times for cinobufagin, resibufogenin, bufalin, bufotalin, arenobufagin and caudatin were 3.07, 3.55, 2.30, 1.62, 1.22 and 3.43 min, respectively. All analytes showed good linearity in a wide concentration range (r  > 0.995) and their lower limits of quantification (LLOQ) were all 1.0 ng/mL. The method was linear for all analytes with correlation coefficients >0.995 for all analytes. The average extract recoveries of the five analytes from rat plasma were all over 85%, the precisions and accuracies determined were all within 15%. This method has been successfully applied to pharmacokinetic study of Chan Su in rats following oral administration.
Keywords: Liquid chromatography tandem mass spectrometry; Chan Su; Bufadienolides; Rat plasma;

A high-throughput analytical method was developed for the measurement of asymmetric dimethylarginine (ADMA) and l-arginine (ARG) from plasma using LC/MS/MS. The sample preparation was simple and only required microfiltration prior to analysis. ADMA and ARG were assayed using mixed-mode ion-exchange chromatography which allowed for the retention of the un-derivatized compounds. The need for chromatographic separation of ADMA from symmetric dimethylarginine (SDMA) was avoided by using an ADMA specific product ion. As a result, the analytical method only required a total run time of 2 min. The method was validated by linearity, with r 2  ≥ 0.995 for both compounds, and accuracy, with no more than 7% deviation from the theoretical value. The estimated limit of detection and limit of quantification were suitable for clinical evaluations. The mean values of plasma ADMA and ARG taken from healthy volunteers (n  = 15) were 0.66 ± 0.12 and 87 ± 35 μM, respectively; the mean molar ratio of ARG to ADMA was 142 ± 81.
Keywords: Asymmetric dimethylarginine; ADMA; l-arginine; ARG; Plasma; LC/MS/MS; Ion-exchange;

Evaluation of sphingolipids changes in brain tissues of rats with pentylenetetrazol-induced kindled seizures using MALDI-TOF-MS by Xiaoqiong Ma; Guangyi Liu; Shuang Wang; Zhong Chen; Maode Lai; Ziyang Liu; Jun Yang (170-177).
Abnormal lipid metabolism has been implicated in the pathogenesis of many neural system diseases, including epilepsy. Pentylenetetrazol (PTZ)-induced kindling in rodents is considered a model of human absence epilepsy and myoclonic, generalized tonic-clonic seizure. In an effort to further understand the mechanism for PTZ-induced seizure, we analyzed crude lipids and sphingolipids in the cortex, hippocampus, and brain stem of normal and PTZ-rats using delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry (DE MALDI-TOF-MS). It was found that phosphatidylcholines dominated the crude lipids in different tissues and there were no obvious differences in crude lipid profiles of different tissues between normal and PTZ-rats. However, ceramide, sphingomyelins, and ceramide-monohexoside were differently distributed in normal and PTZ-rats. Using the reference mass spectra method established in our laboratory, it was shown that sphingomyelins and ceramide-monohexoside levels were elevated in the brain tissues of PTZ-rats. Ceramide levels were found to be higher in brain stem than in cortex and hippocampus of normal rats, and PTZ caused a general decrease in ceramide levels. These data suggest that changes in sphingolipid metabolism contribute to PTZ-induced seizure.
Keywords: Sphingolipids; MALDI-TOF-MS; Epilepsy; Seizure;

A novel HPLC–UV–MS method for quantitative analysis of protein glycosylation by Anton S. Karnoup; Krishna Kuppannan; Scott A. Young (178-191).
Monoclonal antibody samples derived from transgenic plants (plantibodies) may often contain significant amounts of aglycosylated variants. Because glycosylated and non-/de-glycosylated proteins exhibit different functional and pharmacokinetic properties, accurate measurement of non- and de-glycosylated glycoprotein abundances is important. Glycosylation of plant-derived glycoproteins presents specific challenges. Here we describe a novel method to accurately measure relative and absolute amounts of non-glycosylated, de-glycosylated, and total glycosylated protein using an HPLC–UV–MS methodology. Additionally, these results were compared with glycopeptide profiling by MALDI MS. Our studies demonstrated that the quantitative aspect of HPLC–UV method was superior to MALDI MS profiling, which significantly overestimated the relative amounts of aglycosylated species in the isolated glycopeptide fractions.
Keywords: Protein glycosylation; Non-glycosylated; De-glycosylated; Glycoforms; Antibodies; HPLC; UPLC–UV–MS;

Quantification of fat-soluble vitamins in human breast milk by liquid chromatography–tandem mass spectrometry by Maya Kamao; Naoko Tsugawa; Yoshitomo Suhara; Akimori Wada; Toshiyuki Mori; Kazuo Murata; Riichiro Nishino; Tetsuya Ukita; Kazuhiro Uenishi; Kiyoshi Tanaka; Toshio Okano (192-200).
Sensitive quantification method for fat-soluble vitamins in human breast milk by liquid chromatography–tandem mass spectrometry was developed. Vitamins A, D and E were extracted from 10.0 mL of breast milk after saponifying by basic condition. Vitamin K derivatives were extracted from 3.0 mL of breast milk after lipase treatment. The corresponding stable isotope-labeled compounds were used as internal standards. For the determination of vitamin D compounds, derivatization with a Cookson-type reagent was performed. All fat-soluble vitamins were determined by liquid chromatography–tandem mass spectrometry in the positive ion mode. The detection limits of all analytes were 1–250 pg per 50 μL. The recoveries of fat-soluble vitamins were 91–105%. Inter-assay CV values of each vitamin were 1.9–11.9%. The mean concentrations of retinol, vitamin D3, 25-hydroxyvitamin D3, α-tocopherol, phylloquinone and menaquinone-4 were 0.455 μg/mL, 0.088 ng/mL, 0.081 ng/mL, 5.087 μg/mL, 3.771 ng/mL, and 1.795 ng/mL, respectively (n  = 82). This method makes possible to determine fat-soluble vitamins with a wide range of polarities in human breast milk. The assay may be useful for large-scale studies.
Keywords: Vitamin A; Vitamin D; Vitamin E; Vitamin K; Fat-soluble vitamins; Liquid chromatography–tandem mass spectrometry; Breast milk;

The authors have developed a new passive flux sampler (PFS), which was a simple device to determine emission fluxes of potential biomarkers such as acetaldehyde and acetone emanating from the surface of the human skin. The sampler was placed on the skin surface to create a headspace. Within the space, gases emanating from skin moved toward the trapping filter (DNPH impregnated filter) by molecular diffusion and the trapped carbonyls were subsequently determined by HPLC. The PFS was practically applied to volunteers. The emission flux varies with sampling positions, probably depending on the different emanation routes. Personal emission flux also showed great variations between individuals.
Keywords: Skin gas; Acetaldehyde; Acetone; Emission flux; Passive sampler; HPLC;

A quantitative analytical procedure for the determination of cocaine, benzoylecgonine and cocaethylene and norcocaine in hair has been developed and validated. The hair samples were washed, incubated, and any drugs present were quantified using mixed mode solid-phase extraction and liquid chromatography with tandem mass spectrometric detection in positive atmospheric pressure chemical ionization mode. For confirmation, two transitions were monitored and one ion ratio was determined, which was within 20% of that of the known calibration standards. The monitoring of the qualifying transition and requirement for its presence within a specific ratio to the primary ion limited the sensitivity of the assay, particularly for benzoylecgonine, however, the additional confidence in the final result as well as forensic defensibility were considered to be of greater importance. Even with simultaneous monitoring, the concentrations proposed by the United States Federal guidelines for hair analysis were achieved. The limits of quantitation were 50 pg/mg; the limit of detection was 25 pg/mg. The intra-day precision of the assays at 100 pg/mg (n  = 5) was 1.3%, 8.1%, 0.8% and 0.4%; inter-day precision 4.8%, 9.2%, 15.7% and 12.6% (n  = 10) for cocaine, benzoylecgonine, cocaethylene and norcocaine, respectively. The methods were applied to both proficiency specimens and to samples obtained during research studies in the USA.
Keywords: Hair; Cocaine; Benzoylecgonine; Cocaethylene; Norcocaine; LC/MS/MS;

A method for the simultaneous determination of R- and S-propranolol and R- and S-hyoscyamine in human plasma was developed, validated and applied to the analysis of samples from a clinical study. Sample preparation was performed by solid-phase extraction of 2 ml of human plasma using Oasis MCX cartridges and the enantioselective separations were achieved using a Chirobiotic V chiral stationary phase. The chromatography was carried out using gradient elution with a mobile phase composed of methanol:acetic acid:triethylamine which was varied from 100:0.05:0.04 to 100:0.05:0.1 (v/v/v) over 30 min and delivered at a flow rate 1 ml/min. The internal standard was R,S-propranolol-d7 and the analytes were quantified using a single quadrupole mass spectrometer employing APCI interface operated in the positive ion mode with single ion monitoring. The enantioselective separation factors, α, were 1.15 and 1.07 for S- and R-propranolol and R- and S-hyoscyamine, respectively. The standard curves were linear for all target compounds with coefficients of determination (r 2) ranging from 0.9977 to 0.9999. The intra- and inter-day precision and accuracy were ≤13.2% and ≤10.2%, respectively. The assay was used to analyze plasma samples from seven subjects who had received i.v. infusions of R,S-propranolol and R,S-hyoscyamine. The initial data indicate that R-propranolol was eliminated faster than S-propranolol (CL/f  = 2.34 ± 0.13 L/kg min vs. 2.07 ± 0.22 L/kg min) and that R-propranolol had a larger volume of distribution at steady-state (V ss/f  = 705 ± 165 L/kg vs. 589 ± 130 L/kg). In the case of R,S-hyoscyamine, S-hyoscyamine was eliminated faster than R-hyoscyamine (CL/f  = 0.0537 ± 0.0073 L/kg min vs. 0.0439 ± 0.0086 L/kg min), while the volumes of distribution at steady-state were similar for the hyoscyamine enantiomers (V ss/f  = 7.82 ± 2.66 L/kg vs. 7.73 ± 1.39 L/kg).
Keywords: Atropine; Propranolol; Chirobiotic V chiral stationary phase; LC–APCI-MS;

Porcine pancreatic lipase partition in potassium phosphate–polyethylene glycol aqueous two-phase systems by Georgina Bassani; Beatriz Farruggia; Bibiana Nerli; Diana Romanini; Guillermo Picó (222-228).
This research study examined porcine pancreatic lipase partition in aqueous two-phase systems formed by polyethylene glycol-potassium phosphate at pH 6.0, 7.0 and 8.0, the effect of polymer molecular mass, and NaCl concentration. The enzyme was preferentially partitioned into the polyethylene glycol rich phase in systems with molecular mass 4000–8000, while with polyethylene glycol of 10,000 molecular mass it was concentrated in the phosphate rich phase. The enthalpic and entropic changes found due to the protein partition were negative for all the polyethylene glycol molecular mass systems assessed. Both thermodynamic functions were shown to be associated by an entropic–enthalpic compensation effect suggesting that the water structure ordered in the ethylene chain of polyethylene glycol plays a role in the protein partition. The addition of NaCl increased the lipase affinity to the top phase and this effect was most significant in the system polyethylene glycol 2000–NaCl 3%. This system yielded an enzyme recovery more than 90% with a purification factor of approximately 3.4.
Keywords: Lipase; Aqueous two-phase systems; Partition; Polyethyleneglycol;

Determination of beraprost in human plasma by a high-performance liquid chromatography–tandem mass spectrometry by J. Lee; H. Kim; J.C. Jeong; E.S. Park; K.W. Hwang; S.J. Yang; J.H. Jeong (229-233).
A simple, rapid, sensitive and specific liquid chromatography–tandem mass spectrometry method was developed and validated for quantification of beraprost, a stable, orally active prostacyclin analogue with vasodilatory, antiplatelet and cytoprotective effects. The analyte and internal standard, indomethacin, were extracted by solid-phase extraction using OASIS HLB cartridge. The chromatographic separation was performed on a C18 column with a mobile of 0.1% formic acid–methanol (30:70, v/v). The highest daughter ion of deprotonated analyte was quantitated in negative ionization by multiple reactions monitoring with a mass spectrometer. The mass transitions m/z 397 > 269 and m/z 356 > 312 were used to measure beraprost and internal standard, respectively. The assay exhibited a linear range from 0.02 to 2 ng/mL for beraprost in human plasma. The lower limit of quantitation was 20 pg/mL with a relative standard deviation of less than 20%. The method was validated with respect to linearity, sensitivity, specificity, recovery, accuracy and precision. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic study.
Keywords: Beraprost; Prostacyclin; Tandem mass spectrometry; Pharmacokinetics;

HPLC–MS method for the simultaneous quantification of the new HIV protease inhibitor darunavir, and 11 other antiretroviral agents in plasma of HIV-infected patients by Antonio D’Avolio; Marco Siccardi; Mauro Sciandra; Baietto Lorena; Stefano Bonora; Laura Trentini; Giovanni Di Perri (234-240).
A new method using high performance liquid chromatography coupled with electrospray mass spectrometry (HPLC–MS) was developed and validated, for the quantification of plasma concentration of the new protease inhibitors darunavir (DRV) and other 11 antiretroviral agents (ritonavir, amprenavir, atazanavir, lopinavir, saquinavir, indinavir, nelfinavir and its metabolite M-8, nevirapine, efavirenz and tipranavir). A simple protein precipitation extraction procedure was applied on 50 μl of plasma aliquots and chromatographic separation of drugs and Internal Standard (quinoxaline) was achieved with a gradient (acetonitrile and water with formic acid 0.05%) on an C-18 reverse phase analytical column with 25 min of analytical run. Calibration curves were optimised according to expected ranges of drug concentrations in patients, and correlation coefficient (r 2) was higher than 0.998 for all analytes. Mean intra- and inter-day precision (relative standard deviation %) for all compounds were 8.4 and 8.3%, respectively, and mean accuracy (% of deviation from nominal level) was 3.9%. Extraction recovery ranged within 93 and 105% for all drugs analysed. This novel HPLC–MS methodology allows a specific, sensitive and reliable determination of DRV and 11 other antiretrovirals. In our hand, it was used to measure DRV and ritonavir plasma concentration in HIV-positive patients, and it is now successfully applied for routine therapeutic drug monitoring and pharmacokinetics studies.
Keywords: TMC114; DRV; Protease inhibitors; NNRTI; Protein precipitation extraction; HPLC–MS; Quantification;

A sensitive and specific method for the simultaneous determination of the unchanged drug (solifenacin) and its major metabolite (M1, 4S-hydroxy solifenacin) in rat plasma was developed and validated. Both solifenacin and M1 were extracted from rat plasma by a two-step liquid–liquid extraction and analyzed by semi-micro HPLC with UV detection at an absorbance wavelength of 220 nm. The chromatographic separations were performed on a TSKgel ODS-80Ts (5 μm, 150 mm × 2.0 mm i.d.) reversed-phase column with a mobile phase of 0.1 M phosphate buffer (pH 3.0):acetonitrile (71:29, v/v). The intra-day precision (expressed as coefficient of variation, CV) ranged from 0.4% to 1.7%, and the accuracy (expressed as relative error, RE) ranged from −5.2% to 2.0% for solifenacin. The corresponding precision ranged from 1.3% to 3.2%, and accuracy ranged from −4.0% to 8.6% for M1. The lower limit of quantitation for both solifenacin and M1 was 2 ng/ml when 1 ml of plasma was used. No endogenous interference was observed in rat plasma.
Keywords: Solifenacin succinate (YM905); Metabolite; HPLC; UV detection; Rat; Plasma;

Herein two different methods are proposed for the determination of 10 quinolones (enoxacin, ofloxacin, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin, oxolinic acid, nalidixic acid and flumequine) in chicken muscle and egg yolk. Two different HPLC systems were used comparatively and the respective methods were fully validated. The analytes were initially extracted from chicken muscle and egg yolk and purified by a solid phase extraction using LiChrolut RP-18 cartridges. Recoveries varied between 96.6 and 102.8% for chicken muscle and 96.4–102.8% for egg yolk. HPLC separation was performed at 25 °C using an ODS-3 PerfectSil®Target (250 mm × 4 mm) 5 μm analytical column (MZ-Analysentechnik, Germany). The mobile phase consisted of a mixture of 0.1% trifluoroacetic acid (TFA)–ACN–CH3OH, delivered by a gradient program, different for each method. In both cases caffeine was used as internal standard at the concentration of 7.5 ng/μL. Column effluent was monitored using a photodiode array detector, set at 275 and 255 nm. The developed methods were validated according to the criteria of Commission Decision 2002/657/EC. The LODs for chicken muscle varied between 5.0 and 12.0 μg/kg and for egg yolk was 8.0 μg/kg for all examined analytes.
Keywords: Quinolones; Solid phase extraction; Chicken muscle; Egg yolk; HPLC; Commission decision 2002/657/EC;

Quantitation of iptakalim in human plasma by high-performance liquid chromatography–tandem mass spectrometry by Guosheng Teng; Yingwu Wang; Yunbiao Tang; Rui Wang; Yi Fang; J. Paul Fawcett; Jingkai Gu (256-260).
This paper describes a rapid and sensitive analytical method for the quantitation of iptakalim, a novel antihypertensive drug, in human plasma. The method is based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) using sildenafil as internal standard. Sample preparation involved liquid–liquid extraction with dichloromethane–diethyl ether (2:3, v/v) in a basic environment. Chromatography was carried out on an amino column with a mobile phase consisting of acetonitrile–water (55:45, v/v, water containing 0.5% formic acid). Detection employed electrospray ionization (ESI) tandem mass spectrometry in the multiple-reaction-monitoring (MRM) mode. The assay was linear in the concentration range of 0.5–100 ng/ml with a lower limit of quantitation (LLOQ) of 0.5 ng/ml. Intra- and inter-day precision (R.S.D.) were <4.5% and <12.0%, respectively and the accuracy (R.E.) was in the range ±5%. The method was successfully applied to a single oral dose pharmacokinetic study in human volunteers.
Keywords: Iptakalim; LC–MS/MS; Human plasma;

A simple, sensitive, specific and high-resolution reversed-phase liquid chromatographic method utilizing ultraviolet detection has been developed and validated for simultaneous determination of topotecan and four intestinal permeability markers (atenolol, antipyrine, propranolol and furosemide) as suggested by US-FDA. Chromatography was carried out on C-18 column with mobile phase comprising water (pH 3.0) and acetonitrile gradient pumped at a flow rate of 1 ml min−1. The validation parameters included specificity, accuracy, precision, sensitivity and stability studies. Topotecan, an anti-cancer drug widely used in metastatic carcinoma, is a P-glycoprotein substrate having oral bioavailability of 30% with large inter-patient variability. The present method was successfully applied for demonstrating P-gp mediated transport of topotecan and its inhibition using verapamil in Caco-2 cell monolayer. The method can be used in identification of novel P-gp inhibitors for topotecan and estimating the contribution of P-gp in affecting oral bioavailability of topotecan. The other applications of method include its use in validation of Caco-2 monolayer assay for getting biowaiver based on Biopharmaceutic Classification System and its extrapolation to in situ and/or in vivo studies.
Keywords: Topotecan; P-glycoprotein; US-FDA listed intestinal permeability markers; Caco-2;

This paper reports the development of liquid chromatographic columns containing immobilized organic anion transporters (hOAT1 and hOAT2). Cellular membrane fragments from MDCK cells expressing hOAT1 and S2 cells expressing hOAT2 were immobilized on the surface of the immobilized artificial membrane (IAM) liquid chromatographic stationary phase. The resulting stationary phases were characterized by frontal affinity chromatography, using the marker ligand [3H]-adefovir for the hOAT1 and [14C]-p-aminohippurate for the hOAT2 in the presence of multiple displacers. The determined binding affinities (K d) for eight OAT1 ligands and eight OAT2 ligands were correlated with literature values and a statistically significant correlation was obtained for both the hOAT1 and hOAT2 columns: r 2  = 0.688 (p  < 0.05) and r 2  = 0.9967 (p  < 0.0001), respectively. The results indicate that the OAT1 and OAT2 have been successfully immobilized with retention of their binding activity. The use of these columns to identify ligands to the respective transporters will be presented.
Keywords: hOAT1; hOAT2; Drug transporters; Affinity chromatography;

Optimization and validation of a high-performance liquid chromatographic method with UV detection for the determination of pyrrole–imidazole polyamides in rat plasma by Akiko Fukasawa; Takashi Nagashima; Takahiko Aoyama; Noboru Fukuda; Hiroyuki Matsuda; Takahiro Ueno; Hiroshi Sugiyama; Hiroki Nagase; Yoshiaki Matsumoto (272-275).
A simple and sensitive high-performance liquid chromatography (HPLC) method utilizing UV detection was developed for the determination of plasma pyrrole (Py)–imidazole (Im) polyamides in rats and applied to the pharmacokinetic study of compounds. After deproteinization of plasma with methanol, Py–Im polyamides were analyzed with a reversed-phase TSK-GEL ODS-80TM (4.6 mm × 15.0 cm TOSOH Co., Japan) column maintained at 40 °C. The mobile phase solvent A was 0.1% acetic acid and the solvent B was HPLC-grade acetonitrile (0–10 min, A: 100–20%, B: 0–80% linear gradient; 10–15 min, A: 40%, B: 60%). The flow rate was 1.0 ml/min. The detection wavelength was set at 310 nm. The method was used to determine the plasma concentration time profiles of Py–Im polyamides after intravenous injection.
Keywords: Pyrrole–imidazole polyamide; HPLC; Rat plasma; Pharmacokinetics;

Difference in the hydrophilic properties of mycophenolic acid metabolites makes it technically difficult to simultaneously determine their plasma levels in one analytical run. Therapeutic drug monitoring (TDM) for MPA ensures adequate MPA exposure levels to both prevent rejection and avoid related toxicity. One measure limitation for TDM for MPA is the availability of simple, rapid and reproducible method for determination of MPA derivatives.Herein we report a single method to measure MPA and its metabolites using a gradient elution system in less than 10 min. We further tested applicability of our method in both stable and unstable renal transplant recipients with a wide range of levels.Intra- and inter-day imprecision were less than 8% and 10%, respectively. Accuracy of the estimated concentrations ranges from 90% to 108%.Collectively these data show that the new method is reasonably accurate and precise for the simultaneous determination of MPA and its metabolites in human plasma.
Keywords: Pharmacokinetics; Mycophenolic acid; Therapeutic drug monitoring; Transplantation; HPLC assay; Validation;

A new analytical method for simultaneous determination of eight quinolones namely, ciprofloxacin, danofloxacin, difloxacin, enrofloxacin, flumequine, marbofloxacin, oxolinic acid and sarafloxacin, in pig kidney samples was developed. The procedure involves the extraction of the quinolones from the samples by traditional extraction, a step for clean-up and preconcentration of the analytes by solid-phase extraction (SPE) and subsequent liquid chromatography separation with fluorescence detection (LC–FD). The mobile phase was composed of acetonitrile and 10 mM citrate buffer solution of pH 4.5, with an initial composition of acetonitrile–water 12:88 (v/v) and using linear gradient elution. Norfloxacin was used as internal standard. The limits of detection (1–8 μg kg−1) and the limits of quantification (5–27 μg kg−1) found were lower than the maximum residue limits regulated by the European Union for these compounds in pig kidney.
Keywords: Quinolones; Pig kidney; Liquid chromatography–fluorescence detection (LC–FD);