Journal of Chromatography B (v.876, #1)

A highly sensitive and specific HPLC–ESI-MS/MS method has been developed and validated for the estimation of centchroman with 100 μL rat plasma using tamoxifen as an internal standard (IS). The assay procedure involved a single-step, liquid–liquid extraction of centchroman and IS from plasma with 2.5% (v/v) isopropanol in n-hexane, which yielded consistent recoveries of 109.5 and 107.8% for centchroman and IS in rat plasma, respectively. The total chromatographic run time was 3.8 min. Peaks were resolved using 0.01 M ammonium acetate (pH 4.5):acetonitrile (10:90, v/v) mobile phase on a Supelco Discovery C18 column. Specificity and matrix effect on ionization was determined and found that method was specific and there was no significant matrix effect. Linearity range was found to be 0.5–100.0 ng/mL with a correlation coefficient (r) of 0.9959 or better. The intra- and inter-day assay precision ranged from 3.3 to 9.0% and 5.5 to 6.8%, respectively, and intra- and inter-day assay accuracy was between 93.4–107.1% and 96.2–104.2%, respectively. Stability of centchroman in rat plasma was >89.0% in the battery of stability studies viz., bench-top, auto-sampler, freeze/thaw cycles and 30 days storage in a freezer at −80 °C. The assay was successfully applied to determine the pharmacokinetic parameters in SpragueDawley rats after an oral administration of centchroman at 20 mg/kg. As a result, the plasma half-life was 29.4 ± 2.3 h and the AUC(0–∞) was 7345.1 ± 21.9 ng h/mL. The maximum plasma concentration (C max) 117.5 ± 15.7 ng/mL was achieved at 9.0 ± 8.6 h (t max).
Keywords: Centchroman; Rat plasma; Validation; LC–MS/MS; Pharmacokinetics;

Purification of His-tagged proteins using Ni2+–poly(2-acetamidoacrylic acid) hydrogel by Eun-Ju Ha; Yu-Jin Kim; Seong Soo A. An; Young-Rok Kim; Jang-Oo Lee; Sun-Gu Lee; Hyun-jong Paik (8-12).
In this study, a new matrix for immobilized metal affinity chromatography (IMAC) using poly(2-acetamidoacrylic acid) (PAAA) hydrogels complexed with Ni2+ was developed for the purification of the recombinant histidine-tagged green fluorescence protein (His6–GFP). The Ni2+-complexed PAAA hydrogel was prepared by polymerizing 2-acetamidoacrylic acid (AAA) and 2,2′-[(1,4-dioxo-1,4-butanediyl)diamino] bis(2-propenoic acid) (DBDBPA) with potassium persulfate in DMSO, followed by Ni2+ complexation. Confocal laser scanning microscopy was used to determine the binding of His6–GFP to the Ni2+–PAAA hydrogel in three-dimensional space. Photoluminescence spectroscopy revealed an 81% binding efficiency of His6–GFP to the Ni2+–PAAA hydrogel yielded with a recovery of 59%. The specificity of His6–GFP binding to Ni2+–PAAA hydrogel was compared with that of the PAAA hydrogel without Ni2+. His6–GFP was purified directly from the cell lysate with Ni2+–PAAA hydrogel matrix but the PAAA hydrogel without Ni2+ had no effect. The major advantage of the Ni2+–PAAA hydrogel system over current methods, such as Ni–nitrilotriacetic acid (NTA) agarose beads, was the simple and low-cost procedure for preparing the matrix.
Keywords: Poly(2-acetamidoacrylic acid) hydrogel; Immobilized metal affinity chromatography; Protein purification; His-tagged proteins;

Assessment of antibody fragmentation by reversed-phase liquid chromatography and mass spectrometry by Hongcheng Liu; Georgeen Gaza-Bulseco; Edwin Lundell (13-23).
Antibody fragmentation in the hinge region and other regions, and the impact of pH on the level and pattern of antibody fragmentation were investigated by reversed-phase (RP) liquid chromatography and mass spectrometry (LC–MS). Extensive fragmentation was observed in the hinge and in regions other than the hinge of a recombinant monoclonal antibody that was incubated in buffers of various pH at 40 °C for 10 weeks. Peptide bonds that were susceptible to hydrolysis were located mainly around the domain–domain interfaces close to or in the loop structures. The sites as well as the level of peptide bond hydrolysis were affected by the buffer pH. In agreement with previous findings when only the hinge region fragmentation was monitored, pH 6 was optimal for slowing down antibody fragmentation in regions other than the hinge. It also demonstrated that analysis by RPLC–MS provided a better assessment of the susceptible regions of recombinant monoclonal antibodies than size-exclusion chromatography (SEC) followed by fraction collection and mass spectrometry identification.
Keywords: Recombinant monoclonal antibody; Reversed-phase chromatography; Mass spectrometry; Fragmentation;

The work presented here deals with the development of a quantitative tool for the determination of the quaternary ammonium anticholinergic glycopyrrolate in human plasma samples. Mepenzolate was used as an internal standard. The plasma samples were subjected to a suitable sample clean-up consisting of a simple and relatively fast, two step liquid–liquid ion-pair extraction procedure. The chromatography, using the same volatile ion-pair reagent heptafluorobutyric acid (HFBA), takes only 10 min. Relative standard deviation of retention times was never above 2.26% (n  = 36). The method was fully validated based on the US FDA Bioanalytical Method Validation Guidance for Industry. As such, a quantitative ESI-LC–MS(/MS) (TOF mass spectrometry) method was optimized for the absolute quantification of glycopyrrolate in human plasma in a concentration range from 0.101 to 101 ng/mL using a quadratic calibration function (R 2  = 0.9995), y  = −2.21 × 10−4 (±3.93 × 10−5) ×  x 2  + 5.85 × 10−2 (±5.27 × 10−3) ×  x  + 4.08 × 10−3 (±4.82 × 10−4). For the three QC concentrations (QC1 0.252, QC2 2.52, and QC3 25.2 ng/mL) and the LLOQ (0.101 ng/mL), total precision was under 20% (18.0% (n  = 6) at the LLOQ) and maximum accuracy was 112% (88.9% for the LLOQ, n  = 6). Absolute matrix effect (maximum 133% ± 9.59, n  = 3), absolute recovery (better than 41.8% ± 2.22, n  = 3), relative (inter-subject) matrix effect (maximum 10.9% ± 1.45, n  = 4) and process efficiency (better than 45.2% ± 5.74, n  = 3) too were assessed at the 3 QC concentrations.
Keywords: LC–MS/MS; Glycopyrrolate; Pharmacokinetics;

Searching biomarker candidates in serum using multidimensional native chromatography. II Method evaluation with Alport syndrome and severe inflammation by Adrienne Baum; Michael Pohl; Stefan Kreusch; Gerhard A. Cumme; Günter Ditze; Joachim Misselwitz; Michael Kiehntopf; Lene Udby; Andreas Meier-Hellmann; Heidrun Rhode (31-40).
Biomarker search using multidimensional native liquid fractionation of serum in microplates was evaluated. From different donors, homologous sample fractions with UV absorbance depending on state of illness were selected, and their constituents were identified and quantitated by MS. Analysis of sera of patients with Alport syndrome and severe inflammation proved the reliability of the method by confirming characteristic alterations. Moreover, 23 new marker candidates were detected for Alport syndrome, some of them being involved in matrix degradation and repair, and 33 new candidates for severe inflammation, among them α1B-glycoprotein cysteine-rich secretory protein and an apparently low molecular-weight albumin variant.
Keywords: Biomarker search; Multidimensional chromatography; Serum; Alport syndrome; Sepsis; Albumin;

A sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the determination of hydroxysafflor yellow A (HSYA) in human plasma. HSYA was extracted from human plasma by using solid-phase extraction technique. Puerarin was used as the internal standard. A Shim-pack VP-ODS C18 (150 mm × 4.6 mm, 5 μm) column and isocratic elution system composing of methanol and 5 mM ammonium acetate (80:20, v/v) provided chromatographic separation of analytes followed by detection with mass spectrometry. The mass transition ion-pair was followed as m/z 611.19 → 491.19 for HSYA and m/z 415.19 → 295.10 for puerarin. The proposed method has been validated with a linear range of 1–1000 ng/ml for HSYA with a correlation coefficient ≥0.999. The lower limit of quantitation was 1 ng/ml. The intra-batch and inter-batch precision and accuracy were within 10%. The average extraction recovery was 81.7%. The total run time was 5.5 min. The validated method was successfully applied to the study on pharmacokinetics of HSYA in 12 healthy volunteers after a single oral administration of safflower oral solution containing 140 mg of HSYA.
Keywords: Hydroxysafflor yellow A; LC–MS/MS; Human plasma;

Characterization of the in vivo and in vitro metabolic profile of PAC-1 using liquid chromatography–mass spectrometry by Lei Ren; Kaishun Bi; Ping Gong; Weiming Cheng; Zhen Song; Lina Fang; Xiaohui Chen (47-53).
In the present study, the metabolic profile of PAC-1, a potential anticancer drug, was investigated using liquid chromatography–mass spectrometric (LC/MS) techniques. Two different types of mass spectrometers – a quadrupole time-of-flight (Q-TOF) mass spectrometer and an ion trap (IT) mass spectrometer – were employed to acquire structural information on PAC-1 metabolites. A gradient liquid chromatographic system composed of 0.2% formic acid in methanol and 0.2% formic acid in water was used for metabolite separation on an Agilent TC-C18 column. A total of 16 metabolites were detected. The corresponding product ion spectra were acquired and interpreted, and structures were proposed. Accurate mass measurement using LC-Q-TOF was used to determine the elemental composition of metabolites thereby confirming the proposed structures of these metabolites. Phase I metabolic changes were predominantly observed, including debenzylation, dihydrodiol formation, hydroxylation, and dihydroxylation. The detected phase II metabolites included PAC-1 and hydroxylated PAC-1 glucuronide conjugates. Based on metabolite analysis, several PAC-1 metabolic pathways in rat were proposed.
Keywords: Metabolite identification; PAC-1; TOF/MS; Ion trap;

Major pitfalls in the measurement of artemisinin derivatives in plasma in clinical studies by N. Lindegardh; W. Hanpithakpong; B. Kamanikom; P. Singhasivanon; D. Socheat; P. Yi; A.M. Dondorp; R. McGready; F. Nosten; N.J. White; N.P.J. Day (54-60).
A bioanalytical method for the analysis of artesunate (ARS) and its metabolite dihydroartemisinin (DHA) in human plasma using protein precipitation and liquid chromatography coupled to positive tandem mass spectroscopy was developed. The method was validated according to published US FDA-guidelines and showed excellent performance. However, when it was applied to clinical pharmacokinetic studies in malaria, variable degradation of the artemisinins introduced an unacceptable large source of error, rendering the assay useless. Haemolytic products related to sample collection and malaria infection degraded the compounds. Addition of organic solvents during sample processing and even low volume addition of the internal standard in an organic solvent caused degradation. A solid phase extraction method avoiding organic solvents eliminated problems arising from haemolysis induced degradation. Plasma esterases mediated only approximately 20% of ex vivo hydrolysis of ARS into DHA. There are multiple sources of major preventable error in measuring ARS and DHA in plasma samples from clinical trials. These various pitfalls have undoubtedly contributed to the large inter-subject variation in plasma concentration profiles and derived pharmacokinetic parameters for these important antimalarial drugs.
Keywords: Antimalarial; Artesunate; Degradation; Dihydroartemisinin; Liquid chromatography/tandem mass spectrometry (LC/MS/MS); Pitfalls; Protein precipitation; Stable isotope labeled (SIL) internal standard; Solid phase extraction;

A liquid chromatographic-tandem mass spectrometric method for determination of artesunate and its metabolite dihydroartemisinin in human plasma by W. Hanpithakpong; B. Kamanikom; A.M. Dondorp; P. Singhasivanon; N.J. White; N.P.J. Day; N. Lindegardh (61-68).
A bioanalytical method for the analysis of artesunate and its metabolite dihydroartemisinin in human plasma using high throughput solid-phase extraction in the 96-wellplate format and liquid chromatography coupled to positive tandem mass spectroscopy has been developed and validated. The method was validated according to published FDA guidelines and showed excellent performance. The within-day and between-day precisions expressed as RSD, were lower than 7% at all tested concentrations including the lower limit of quantification. Using 50 μl plasma the calibration range was 1.19–728 ng/ml with a limit of detection at 0.5 ng/ml for artesunate and 1.96–2500 ng/ml with a limit of detection at 0.6 ng/ml for dihydroartemisinin. Using 250 μl of plasma sample the lower limit of quantification was decreased to 0.119 ng/ml for artesunate and 0.196 ng/ml dihydroartemisinin. Validation of over-curve samples in plasma ensured that accurate estimation would be possible with dilution if samples went outside the calibration range. The method was free from matrix effects as demonstrated both graphically and quantitatively.
Keywords: Antimalarial; Artesunate; Dihydroartemisinin; High throughput; Liquid chromatography/tandem mass spectrometry (LC-MS/MS); Stable isotope labeled (SIL) internal standard; Solid phase extraction (SPE);

Studies of verapamil binding to human serum albumin by high-performance affinity chromatography by Rangan Mallik; Michelle J. Yoo; Sike Chen; David S. Hage (69-75).
The binding of verapamil to the protein human serum albumin (HSA) was examined by using high-performance affinity chromatography. Many previous reports have investigated the binding of verapamil with HSA, but the exact strength and nature of this interaction (e.g. the number and location of binding sites) is still unclear. In this study, frontal analysis indicated that at least one major binding site was present for R- and S-verapamil on HSA, with estimated association equilibrium constants on the order of 104  M−1 and a 1.4-fold difference in these values for the verapamil enantiomers at pH 7.4 and 37 °C. The presence of a second, weaker group of binding sites on HSA was also suggested by these results. Competitive binding studies using zonal elution were carried out between verapamil and various probe compounds that have known interactions with several major and minor sites on HSA. R/S-Verapamil was found to have direct competition with S-warfarin, indicating that verapamil was binding to Sudlow site I (i.e. the warfarin–azapropazone site of HSA). The average association equilibrium constant for R- and S-verapamil at this site was 1.4 (±0.1) × 104  M−1. Verapamil did not have any notable binding to Sudlow site II of HSA but did appear to have some weak allosteric interactions with l-tryptophan, a probe for this site. An allosteric interaction between verapamil and tamoxifen (a probe for the tamoxifen site) was also noted, which was consistent with the binding of verapamil at Sudlow site I. No interaction was seen between verapamil and digitoxin, a probe for the digitoxin site of HSA. These results gave good agreement with previous observations made in the literature and help provide a more detailed description of how verapamil is transported in blood and of how it may interact with other drugs in the body.
Keywords: Verapamil; Human serum albumin; Drug–protein binding; High-performance affinity chromatography;

Determination of 4-dimethylaminophenol concentrations in dog blood using LC–ESI/MS/MS combined with precolumn derivatization by Xiao Mei Zhuang; Mei Yuan; Zhi Wei Zhang; Xiao Ying Wang; Zhen Qing Zhang; Jin Xiu Ruan (76-82).
A sensitive and reproducible LC–ESI/MS/MS method, which was combined with the precolumn dansyl chloride derivatization to enhance the signal intensity of analytes, was developed to determine blood 4-dimethylaminophenol (DMAP) concentrations. The linearity of the method was observed within the concentration range of 2–2000 ng/mL. The precision, accuracy, stability, recovery and matrix effect of the method were also investigated and found to meet the requirements for pharmacokinetic studies of the drug. By using this method, pharmacokinetic studies were conducted in dogs after i.m. and i.v. administrations. The results showed that DMAP could not only be absorbed into blood quickly after i.m., but also can be eliminated rapidly. Both i.m. and i.v. routes are appropriate for DMAP to be used in field first-aid. It has been proved that this LC–MS/MS combined with precolumn derivatization method can be used as a routine analytical method to provide enhanced measurements for blood DMAP concentrations. It is also useful for DMAP pharmacokinetic evaluation.
Keywords: DMAP; LC–MS/MS; Precolumn derivatization;

Determination of epoxyeicosatrienoic acids in human red blood cells and plasma by GC/MS in the NICI mode by Sophie Goulitquer; Yvonne Dréano; François Berthou; Laurent Corcos; Danièle Lucas (83-88).
Epoxyeicosatrienoic acids (EETs) are cytochrome P450 metabolites of arachidonic acid involved in the regulation of vascular tone. Despite the importance of EETs in a variety of physiological effects, few methods have been developed to quantify them in human blood. This led us to develop a method by GC/MS with negative ion chemical ionization. As EETs are primarily located in phospholipids, red blood cells (RBCs) and plasma phospholipids were hydrolyzed with phospholipase A2 after a solid phase extraction. Then, EETs were derivatized as pentafluorobenzyl esters, and [2H8]-arachidonic acid was used as internal standard for quantification. EETs were found to be at concentrations of 106 ± 37 ng mL−1 in plasma and 33.4 ± 8.5 ng/109 RBCs (mean ± S.D.) in 10 healthy volunteers. Their amount in RBCs was 3-fold that in plasma; both parameters proved to be well correlated.
Keywords: Epoxyeicosatrienoic acids; Eicosanoids; Cytochrome P450; Human red blood cells; GC/MS/NICI;

Sensitive chiral high-performance liquid chromatographic determination of anthelmintic flubendazole and its phase I metabolites in blood plasma using UV photodiode-array and fluorescence detection by Milan Nobilis; Zuzana Vybíralová; Veronika Křížová; Vladimír Kubíček; Marie Soukupová; Jiří Lamka; Barbora Szotáková; Lenka Skálová (89-96).
Although benzimidazole anthelmintic flubendazole, methyl ester of [5-(4-fluorobenzoyl)-1H-benzimidazol-2-yl]carbamic acid, is extensively used in veterinary and human medicine for the treatment of gastrointestinal parasitic helmint infections, reliable data about its pharmacokinetics in various species have not been reported. Our previous work [M. Nobilis, Th. Jira, M. Lísa, M. Holčapek, B. Szotáková, J. Lamka, L.Skálová, J. Chromatogr. A 1149 (2007) 112–120] had described the stereospecificity of carbonyl reduction during phase I metabolic experiments in vitro. For in vivo pharmacokinetic studies, further improvement and optimization of bioanalytical HPLC method in terms of sensitivity and selectivity was necessary. Hence, a modified chiral bioanalytical HPLC method involving both UV photodiode-array and fluorescence detection for the determination of flubendazole, both enantiomers of reduced flubendazole and hydrolyzed flubendazole in the extracts from plasma samples was tested and validated. Albendazole was used as an internal standard. Sample preparation process involved a pH-dependent extraction of the analytes from the blood plasma into tert-butylmethyl ether. Chromatographic separations were performed on a Chiralcel OD-R 250 mm × 4.6 mm column with mobile phase methanol-1 M NaClO4 (75:25, v/v) at the flow rate 0.5 ml min−1. In quantitation, selective UV absorption maxima of 290 nm (for reduced flubendazole), 295 nm (for albendazole), 310 nm (for flubendazole) and 330 nm (for hydrolyzed flubendazole) were used in the UV photodiode-array detection, and λ(exc.)/λ(emis.) = 228 nm/310 nm (for reduced flubendazole) and λ(exc.)/λ(emis.) = 236 nm/346 nm (for albendazole) were set on the fluorescence detector. The fluorescence detection was approximately 10-times more sensitive than the UV detection. Each HPLC run lasted 27 min. The validated chiral HPLC-PDA-FL method was employed in the pharmacokinetic studies of flubendazole in sheep. The stereospecificity of the enzymatic carbonyl reduction of flubendazole was also observed in vivo. (+)-Reduced flubendazole was found to be the principal metabolite in ovine blood plasma and only low concentrations of hydrolyzed flubendazole, the parent flubendazole and (−)-reduced flubendazole were detected in this biomatrix.
Keywords: Benzimidazole anthelmintics; Phase I flubendazole metabolites; Pharmacokinetics in sheep; Chiral HPLC with UV photodiode-array and fluorescence detection; Stereospecificity of the carbonyl reduction of flubendazole;

In this study, we examined the feasibility of a novel liquid chromatography technique that combines temperature-responsive polymeric materials with inductive heating via an alternating magnetic field (AMF). We considered the following components of the technique: (i) the preparation of composite materials of magnetite and silica, (ii) their heating behavior under the AMF, (iii) the conjugation of temperature-responsive polymers for the packing materials, and (iv) the elution profiles of the model compounds in the AMF. The results showed that we could influence the elution of the model compounds by AMF induction heating generated by the surrounding coil.
Keywords: Thermo-responsive polymer; Poly(N-isopropylacrylamide); Magnetic particle; Magnetic field inductive heating; Liquid chromatography; Packing material;

Simultaneous determination of two acute poisoning rodenticides tetramine and fluoroacetamide with a coupled column in poisoning cases by Xiaomin Xu; Guoliang Song; Yan Zhu; Jing Zhang; Yongxin Zhao; Haitao Shen; Zengxuan Cai; Jianlong Han; Yiping Ren (103-108).
A coupled column system was developed for the simultaneous determination of both rodenticides fluoroacetamide and tetramine in this paper by gas chromatography/mass spectrometry (GC/MS). A short length of strong polar column (1.5 m of Innowax) was coupled to the top of a 30 m of DB-5ms with a quartz capillary column connector. Peak width at half height (W h) was used to evaluate the band broadening of the coupled column system. The length of the short couple column and oven temperature program were discussed according to W h. The precisions of the coupled column were analyzed with peak area and retention time. Good linear correlations were found for both rodenticides. Typical samples were discussed for each rodenticide and some poisoning cases were presented.
Keywords: Rodenticide; Tetramine and fluoroacetamide; Gas chromatography/mass spectrometry; Coupled column; Peak width at half height; Poisoning cases;

Chromatographic purification of equine immunoglobulin G F(ab)2 from plasma by Aldon Fernandes; John Oswald Kaundinya; Gautam Daftary; Lalit Saxena; Subhasis Banerjee; Priyabrata Pattnaik (109-115).
The antibody fragments generated from hyperimmune equine IgG is widely used as anti-snake venom, anti-scorpion venom, anti-diphtheria, anti-tetanus, anti-gangrene and anti-rabies agents. Antibody fragments, F(ab)2, because of their specificity and absence of undesired reactivity are preferred over complete IgG. This paper discusses a novel purification technique for chromatographic purification of anti-rabies immunoglobulin G (IgG) fragment F(ab)2 from horse serum. F(ab)2 was purified by two successive chromatography steps using Cellufine A-200 and ProSep-vA Ultra media. The purified F(ab)2 was characterized using biochemical and biophysical methods and shown to be pure and homogeneous. The purified F(ab)2 was reactive to rabies antigen in immuno-electrophoresis and diffusion tests. The purified F(ab)2 was biologically functional and was found to show a potency of 1500 IU ml−1. Comparative analysis of the purity with commercially available F(ab)2 by HPLC analysis and SDS–PAGE indicated that the present product is better in purity. To our knowledge, this is the first report providing evidence on purification of equine antibody fragment using controlled pore glass based protein A chromatography media.
Keywords: Plasma fractionation; Equine; F(ab)2; Plasma IgG; Purification; Chromatography; Enzymatic digestion; ProSep-vA Ultra; Cellufine A-200;

Adsorption of histidine-containing dipeptides on copper(II) immobilized chelating resin from saline solution by Tatsuya Oshima; Kenzo Kanemaru; Hodzumi Tachiyama; Kaoru Ohe; Yoshinari Baba (116-122).
Adsorption of histidine-containing dipeptides such as carnosine (Car) was investigated using copper(II) immobilized cation exchange resins. Adsorption of Car was enhanced using Cu(II) immobilized resins, on the basis of metal affinity interactions. In particular, iminodiacetic acid chelating resin with immobilized Cu(II) (Cu–IDA) can adsorb Car from saline water. Car was adsorbed on Cu–IDA even in the presence of 1000 mM of NaCl. Adsorption of various amino acids on Cu–IDA was compared under same conditions. Histidine and the histidine-containing dipeptides were selectively adsorbed on Cu–IDA over other amino acids, both in the absence and in the presence of NaCl. Therefore, immobilized metal affinity adsorption is an efficient method for recovering histidine-containing dipeptides from saline water.
Keywords: IMAC (immobilized metal affinity chromatography); Histidine-containing dipeptide; Carnosine; Anserine; Adsorption; Separation;

Quantitative analysis of 5′-deoxy-5′-methylthioadenosine in melanoma cells by liquid chromatography-stable isotope ratio tandem mass spectrometry by Axel P. Stevens; Katja Dettmer; Susanne Wallner; Anja K. Bosserhoff; Peter J. Oefner (123-128).
The frequent deletion of the human chromosomal region 9p21, including the methylthioadenosine phosphorylase (MTAP) gene, is hypothesized to lead to the intra- and/or extracellular accumulation of 5′-deoxy-5′-methylthioadenosine (MTA) in cancer cells and the subsequent promotion of tumor progression. The lack of sensitive methodology for the direct measurement of MTA in tumor cells has hampered the testing of this hypothesis to date. A liquid chromatography electrospray ionization tandem mass spectrometry method (LC–MS/MS) was developed for the absolute quantitative determination of MTA in cell culture media and cell extracts using stable isotope labeled MTA as an internal standard. Limit of detection (LOD) and lower limit of quantification (LLOQ) were 62.5 pM and 2 nM, respectively, and allowed the direct measurement of MTA in biological samples without prior enrichment. Average imprecision of MTA extraction from cells and cell media, as well as LC–MS/MS analysis were 9.7, 3.8 and 1.9%, respectively. The method enabled the demonstration of the accumulation of MTA in melanoma cell culture media reaching a steady-state level within 24 h. Only a slight difference in extracellular MTA concentrations was observed between cells with and without MTAP expression. However, there was a fourfold increase in intracellular MTA concentration in melanoma cells lacking MTAP, thus confirming the hypothesized accumulation of MTA in human cancer cells harboring a chromosome 9p21 deletion.
Keywords: 5′-Deoxy-5′-methylthioadenosine; Methylthioadenosine phosphorylase; LC–MS/MS; Stable isotope dilution; Melanoma;

This manuscript describes the determination of oseltamivir (OP) and oseltamivir carboxylate (OC) in rat plasma, cerebrospinal fluid (CSF) and brain and in human plasma and urine using liquid chromatography coupled to tandem mass spectrometry. Threefold deuterated OP and OC served as internal standards. Protein precipitation with perchloric acid was followed by on-line solid-phase extraction and gradient separation on a reversed-phase column. After electrospray ionization, the compounds were detected in positive ion selected reaction monitoring (SRM) mode. Run time was 3.6 min. The lower limits of quantification (LLOQ) were 0.1 ng/mL in rat plasma and CSF, 0.5 ng/g in brain and 1 ng/mL in human plasma and urine. Inter-day and intra-day precisions and inaccuracies in rat matrices were below 10.2% and 13.9% (below 19.0% at LLOQ), respectively. Intra-assay precisions and inaccuracies in human matrices were below 11.7% and 8.9%, respectively. The recoveries were close to 100%, and no significant matrix effect was observed. The method was successfully applied to rat study samples.
Keywords: Oseltamivir; Oseltamivir carboxylate; Plasma; Brain; Urine; Liquid chromatography–tandem mass spectrometry; On-line solid-phase extraction;

Detection of ketamine and its metabolites in urine by ultra high pressure liquid chromatography–tandem mass spectrometry by Mark C. Parkin; Sophie C. Turfus; Norman W. Smith; John M. Halket; Robin A. Braithwaite; Simon P. Elliott; M. David Osselton; David A. Cowan; Andrew T. Kicman (137-142).
Current analytical methods used for screening drugs and their metabolites in biological samples from victims of drug-facilitated sexual assault (DFSA) or other vulnerable groups can lack sufficient sensitivity. The application of liquid chromatography, employing small particle sizes, with tandem mass spectrometry (MS/MS) is likely to offer the sensitivity required for detecting candidate drugs and/or their metabolites in urine, as demonstrated here for ketamine. Ultra-performance liquid chromatography–mass spectrometry (UPLC–MS/MS) was performed following extraction of urine (4 mL) using mixed-mode (cation and C8) solid-phase cartridges. Only 20 μL of the 250 μL extract was injected, leaving sufficient volume for other assays important in DFSA cases. Three ion transitions were chosen for confirmatory purposes. As ketamine and norketamine (including their stable isotopes) are available as reference standards, the assay was additionally validated for quantification purposes to study elimination of the drug and primary metabolite following a small oral dose of ketamine (50 mg) in 6 volunteers. Dehydronorketamine, a secondary metabolite, was also analyzed qualitatively to determine whether monitoring could improve retrospective detection of administration. The detection limit for ketamine and norketamine was 0.03 ng/mL and 0.05 ng/mL, respectively, and these compounds could be confirmed in urine for up to 5 and 6 days, respectively. Dehydronorketamine was confirmed up to 10 days, providing a very broad window of detection.
Keywords: Ketamine; Metabolites; Drug facilitated sexual assault; Liquid chromatography; Mass spectrometry;

A sensitive and specific method using a one-step liquid–liquid extraction (LLE) with ethyl acetate followed by high-performance liquid chromatography (HPLC) coupled with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection was developed and validated for the determination of roxatidine in human plasma using famotidine as an internal standard (IS). Data acquisition was carried out in multiple reaction monitoring (MRM) mode, by monitoring the transitions m/z 307.3 → 107.1 for roxatidine and m/z 338.4 → 189.1 for famotidine. Chromatographic separation was performed on a reverse phase Hydrosphere C18 column at 0.2 mL min−1 using a mixture of methanol–ammonium formate buffer as mobile phase (20:80, v/v; adjusted to pH 3.9 with formic acid). The achieved lower limit of quantification (LLOQ) was 1.0 ng mL−1 and the standard calibration curve for roxatidine was linear (r 2  = 0.998) over the studied range (1–1000 ng mL−1) with acceptable accuracy and precision. Roxatidine was found to be stable in human plasma samples under short-, long-term storage and processing conditions. The developed method was validated and successfully applied to the bioequivalence study of roxatidine administrated as a single oral dose (75 mg as roxatidine acetate hydrochloride) to healthy female Korean volunteers.
Keywords: Roxatidine; Liquid chromatography–tandem mass spectrometry; Liquid–liquid extraction; Human plasma; Bioequivalence;

A simple, rapid and specific ion-pair liquid chromatographic method for the routine determination of the marker residue of oxytetracycline in sheep milk, at levels as low as 20 μg/kg, has been developed. Milk samples were acidified and extracted with acetonitrile. The extracts were purified by treatment with ammonium sulphate and concentrated into diluted phosphoric acid. Separation was carried out isocratically on a Nucleosil C18 column using a mobile phase that contained both positively and negatively charged pairing ions. The in-house validated method gave overall recoveries and overall relative standard deviations better than 86% and 4.6%, respectively. The method was successfully applied to study the depletion of oxytetracycline in sheep milk and to estimate the withdrawal period after intramuscular administration of a commercial oxytetracycline formulation.
Keywords: Oxytetracycline; 4-epi-Oxytetracycline; Residues; Milk; Ion-pair liquid chromatography; Depletion;