Journal of Chromatography B (v.877, #30)

We developed a rapid, specific and sensitive method based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC–MS/MS) to determine icotinib concentrations in human plasma and urine. Liquid–liquid extraction (LLE) and direct dilution were firstly used to isolate icotinib from plasma and urine followed by injection of the extracts onto a C18 column with gradient elution. Ionization of icotinib and midazolam (internal standard, IS) was performed using an electrospray ionization source in positive mode and detection was carried out in multi-reaction monitoring (MRM) mode. The lower limits of quantitation (LLoQ) of icotinib in human plasma and urine by this method were 0.1 and 1.00 ng/mL, respectively. The accuracy, precision, specificity, recovery, matrix effect, linearity and several of stabilities have been validated for icotinib in human plasma and urine. In conclusion, the validation results showed that this method is robust, specific and sensitive and it can successfully fulfill the requirement of clinical pharmacokinetic study of icotinib hydrochloride in Chinese healthy subjects.
Keywords: Quantitative determination; Icotinib; Human plasma and urine;

A robust and validated liquid chromatography–tandem mass spectrometry (LC–MS/MS) method with positive electrospray ionization (ESI) was developed for the determination of hydrochloride meptazinol in human plasma. After liquid–liquid extraction of 200 μl of human plasma, HPLC separation was achieved on a Thermo Hypurity Cyano column, using acetonitrile:ammonium formate (50 mM, aq) (70:30, v/v) as the mobile phase. The mass spectrometer was operated in multiple reaction monitoring (MRM) mode using the transition m/z 234 → 234 for meptazinol and m/z 152 → 110 for the IS (acetaminophen), respectively. The calibration curves were linear over the range of 0.2925–292.5 ng/ml, with the limit of quantification (LOQ) 0.2925 ng/ml. The mean absolute recovery of hydrochloride meptazinol was more than 70.21%. Intra- and inter-day precisions were less than 9.05% and 12.89%, respectively. And the accuracy was within −2.99% to 4.96%. This method was applied to a pharmacokinetic study of hydrochloride meptazinol tablets in healthy Chinese volunteers.
Keywords: Hydrochloride meptazinol; LC–MS/MS; Pharmacokinetic;

Determination of paromomycin residues in turkey tissues by liquid chromatography/mass spectrometry by K. Róna; G. Klausz; E. Keller; M. Szakay; P. Laczay; M. Shem-Tov; P. Székely-Körmöczy (3792-3798).
A liquid chromatography–electrospray-mass spectrometry method has been developed and validated for the determination of paromomycin in turkey muscle, liver and kidney, using kanamycin as internal standard. The method consists of solid-phase extractions on mixed-mode columns. The chromatographic separation was carried out on a C18 column using binary gradient elution containing acetonitrile and 5 mM pentafluoropropionic-acid in water. The method was evaluated for specificity, linearity, recovery, accuracy, limit of detection, limit of quantification, intra- and inter-day repeatability, and stability. It was proven to be selective, linear, precise and accurate over the concentration range tested (0.5 × MRL–2 × MRL for each tissue) with correlation coefficients >0.990. The method was successfully used for the residue determination of PARO from edible tissues of turkeys.
Keywords: Paromomycin; Turkey; Solid-phase extraction; Liquid chromatography/mass spectrometry;

This is the first report that combines cascade biomimetic affinity fractionation with MS-based proteomics analysis. Our lab has constructed an affinity ligand library composed of thousands of ligands with different protein-binding properties. Structural differences between these ligands result in different non-bonded protein–ligand interactions, thus each ligand exhibits a specific affinity to some protein groups. In this work, we first screened out three affinity ligands with large difference in protein-binding properties. Next, cascade combination of these ligands was applied to fractionate tissue sample into simple subgroups prior to trypsin digestion and LC–MS/MS analysis. In this study, 391 non-redundant protein groups were identified in unfractionated rat liver cytosol, 499 protein groups were identified in 2 fractions of the first affinity fractionation, 616 in 4 fractions of the second fractionation, and 738 in 8 fractions of the third fractionation (an 88.74% increase). Ultimately, a total of 859 unique protein groups were identified in all cascade fractions (a 119.6% increase compared with unfractionated sample). The proteins detected in each fraction were bioinformatically categorized according to their physicochemical characteristics (relative molecular mass, pI, GRAVY value and TM helices). This approach highlighted the sensitivity of this method to a wide variety of protein classes.
Keywords: Biomimetic affinity chromatography; Cascade affinity fractionation; LC–MS/MS; Proteomics; Rat liver cytosol;

A metabonomic approach identifies human urinary phenylacetylglutamine as a novel marker of interstitial cystitis by Yousuke Fukui; Masao Kato; Yohji Inoue; Akio Matsubara; Kohji Itoh (3806-3812).
An ultra-performance liquid chromatography–mass spectrometry (UPLC–MS) based metabonomic approach was applied to identify a candidate metabolite with not known to be associated with interstitial cystitis (IC). IC is a chronic clinical syndrome associated with urinary frequency and urgency and/or pelvic pain. The ability to non-invasively diagnose the early stage of IC would be important for improving the patient's quality of life. The current standard IC diagnosis is cystoscopy, which is invasive and painful. Urine samples from the following were taken and analyzed: 10 IC patients, 10 bacterial cystitis (BC) patients, and 10 healthy volunteers (HVs) to identify an IC marker; and subsequently analyzed 5 IC patients and 5 HVs for marker validation. The urinary marker of IC was identified as phenylacetylglutamine (PAGN) using NMR and MS/MS analysis. In addition, quantitative methods were developed to determining the urinary PAGN levels using UPLC-UV. The urinary level of PAGN measured relative to creatinine (Cr) was significantly elevated in IC patients (mean 0.47 mg/mg Cr) compared with BC patients (mean 0.25 mg/mg Cr) and HVs (mean 0.11 mg/mg Cr). Interestingly, urinary PAGN/Cr ratios in patients with mild IC (grade I) and moderate IC (grade II) were higher than for patients with severe IC (grade III). Moreover, urinary PAGN/Cr ratios with mild and moderate IC patients (mean 0.30 mg/mg Cr) were higher than HVs (mean 0.059 mg/mg Cr), in the validation set. These findings establish urinary PAGN/Cr ratios as a novel urinary marker of IC, and may contribute to early diagnosis of IC patients.
Keywords: Metabonomics; Phenylacetylglutamine; Interstitial cystitis;

This study details the development and validation of a simple and sensitive liquid chromatography/tandem mass spectrometry (LC–MS/MS) method for the quantification of combretastatin A-4 3-O-phosphate (CA4P), combretastatin A4 (CA4) and its main metabolite, combretastatin A4 glucuronide (CA4G), in beagle dog plasma. Sample pretreatment includes simple protein precipitation by adding methanol to the plasma sample containing an internal standard (colchicine). LC separation was successfully accomplished on a Waters RP8 Symmetryshield™ column (3.0 mm×150 mm, i.d., 5 μm) with a gradient mobile phase of methanol (0.1% formic acid, v/v) and water (20 mM ammonium acetate) at a flow rate 0.8 mL min−1. The three analytes were detected in the positive/negative ion alternate mode, negative ion mode for CA4G and positive ion mode for CA4P and CA4. Multiple reaction monitoring (MRM) was used for determination of three analytes. Calibration curves were linear in the concentration range of 5–5000 ng mL−1 for CA4P (r  ≥ 0.999), 5–3000 ng mL−1 for CA4 (r  ≥ 0.999) and 5–5000 ng mL−1 for CA4G (r  ≥ 0.999). All the validation data, such as accuracy, precision, and inter-day repeatability, were within the required limits. The method was reliable and has been successfully applied to a pharmacokinetic study of CA4P in beagle dogs via intravenous drop infusion at dose rates of 1, 3 and 9 mg kg−1. After daily intravenous drop infusions at 1 mg kg−1 for 7 consecutive days, the accumulation ratio was approximately 1.0, indicating no accumulation from multiple doses in beagles.
Keywords: Combretastatin A4 phosphate; Combretastatin A4; Combretastatin A4 glucuronide; LC–MS/MS; Pharmacokinetics;

Separation of microcystins and nodularins by ultra performance liquid chromatography by Lisa Spoof; Milla-Riina Neffling; Jussi Meriluoto (3822-3830).
Four ultra performance liquid chromatography (UPLC) columns with different reversed-phase characteristics were tested in the chromatographic separation of 10 microcystins and three nodularins, cyanobacterial peptide toxins. The columns had been designed by the manufacturer to withstand the ultra-high pressure generated by sub-2 μm stationary phase particles and the Waters ACQUITY UPLC system in ultra-fast separations. The gradient mobile phase consisted of water and acetonitrile, both acidified with trifluoroacetic acid, with three gradient rise times: 1, 1.5 and 2 min. The UV detection of the toxins was performed by a photodiode array detector. The chromatographic performance was evaluated both visually and by calculating chromatographic parameters such as capacity factor, resolution, peak width at half height, selectivity and peak asymmetry. The best chromatographic performance as judged by visual inspection was given by the ACQUITY BEH Shield RP18 and ACQUITY BEH Phenyl columns. The BEH Shield RP18 column showed excellent selectivity and resolution of chosen peak pairs considered as critical. A further advantage of the UPLC system was the high sample throughput with a total analysis time of 3.12 min (injection-to-injection) equalling to 461 separations per 24 h.
Keywords: Ultra performance liquid chromatography (UPLC); Sub-2 μm particles; Reversed-phase; Microcystins; Nodularins; Cyanobacteria; Toxins;

Simultaneous analysis of eight nucleoside triphosphates in cell lines by liquid chromatography coupled with tandem mass spectrometry by Sabine Cohen; Mehdi Megherbi; Lars Petter Jordheim; Isabelle Lefebvre; Christian Perigaud; Charles Dumontet; Jérôme Guitton (3831-3840).
In this study, we developed a new method for the simultaneous determination of eight endogenous ribonucleoside triphosphates and deoxyribonucleoside triphosphates based on a combination of a selective sample preparation and an ion-pair liquid chromatography–electrospray tandem mass spectrometry. The sample preparation was based on a protein precipitation coupled with a solid phase extraction using a weak-anion-exchange cartridge. The analytical separation of the nucleotides was achieved on a porous graphitic carbon stationary phase with a binary elution gradient program employing ion-pairing reagents (diethylamine and hexylamine) and organic eluent (methanol). The triple quadrupole mass spectrometer operated in both negative and positive multiple reaction monitoring modes. The calibration assay used the stable isotope labelled analogs of each compounds as standard. Standard calibrations were from 0.25 to 10 pmol injected according to deoxyribonucleotides and from 12.5 to 3000 pmol injected according to ribonucleotides. The within-run precision of the assay was less than 14.5% and the between-run precision was less than 12.4% for each analytes. Assay accuracy was in the range of 92.3–107.6%. This method allows the determination of NTP and dNTP pools from lysats of several cell lines or peripheral blood mononuclear cell from patient. Assays were performed with different preparation of cells to confirm the quality and the relevance of the described method.
Keywords: Nucleoside triphosphates; Ribonucleosides; Deoxyribonucleosides; Mass spectrometry; Liquid chromatography;

Isomerization plays a key role in protein degradation. This isomerization is often difficult to detect by many protein characterization methods such as SDS-PAGE, SEC, and IEF. This work shows the identification of an isomerized aspartic acid residue in the CDR2 of the heavy chain of a fully human monoclonal antibody. This isoaspartic acid increases significantly with storage at 2–8 °C. Hydrophobic interaction chromatography was utilized to separate the isoaspartic variant in the intact state. Mass spectrometry including peptide mapping was employed to identify and confirm the exact location of the modification. Since this modification occurs in the complementarity determining region (CDR) it was found that binding is reduced. Therefore, three different analytical methods for regular analysis of this isomerization are evaluated. These methods include peptide mapping by LC–MS, HIC, and a protein isoaspartate methyltransferase assay. It was determined that HIC is the best method to regularly assay the level of isomerization in this monoclonal antibody.
Keywords: Monoclonal antibody; Isomerization; Peptide mapping; PIMT assay; Hydrophobic interaction chromatography;

Direct injection liquid chromatography/positive ion electrospray ionization mass spectrometric quantification of methotrexate, folinic acid, folic acid and ondansetron in human serum by Panagiotis Koufopantelis; Sophia Georgakakou; Michael Kazanis; Costas Giaginis; Alexandra Margeli; Sophia Papargiri; Irene Panderi (3850-3856).
A rapid liquid chromatography/positive ion electrospray mass spectrometric assay (LC/ESI-MS) was developed for the quantitation of methotrexate, folinic acid, folic acid and ondansetron in human serum. The assay was based on 100 μL serum samples, following acetonitrile precipitation of proteins and filtration that enabled direct injection into the LC/MS system. All analytes and the internal standard, alfuzosin, were separated by using a Zorbax Eclipse XDB–C8 analytical column (2.1 mm × 150.0 mm i.d., particle size 3.5 μm) with isocratic elution. The mobile phase was composed of a mixture of water/acetonitrile containing 0.1%, v/v formic acid (75:25, v/v), pumped at a flow rate of 0.15 mL min−1. Quantitation of the analytes was performed with selected ion monitoring (SIM) in positive ionization mode using electrospray ionization interface. The assay was found to be linear in the concentration range of 0.01–25.00 μg mL−1 for methotrexate and 0.01–5.00 μg mL−1 for folic acid, folinic acid and ondansetron. Intermediate precision was found to be less than 4.2% over the tested concentration ranges. A run time of less than 7.0 min for each sample made it possible to analyze a large number of human serum samples per day. The method can be used to quantify methotrexate, folinic acid, folic acid and ondansetron in human serum covering a variety of clinical studies and it was applied to the analysis of human serum samples obtained from children with acute lymphoblastic leukemia.
Keywords: Liquid chromatography/mass spectrometry; Methotrexate; Folinic acid; Folic acid; Ondansetron; Human serum;

Described herein are two general screening procedures for the simultaneous determination of 49 exogenous compounds (21 diuretics, 19 beta-blockers, eight stimulants and two steroidal drugs) in human urine by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Urine samples were extracted using a simple and robust solid phase extraction (SPE) method. Samples were injected onto reversed phase HPLC and UPLC columns connected to tandem mass spectrometers capable of scan-to-scan polarity switching. The methods were validated according to the ISO 17025 international standard for the validation of a qualitative method. Sixty urine samples submitted for routine analysis were tested using both methods, the results of which correlated with results obtained from previously validated procedures. Both methods proved to be useful for routine urine analysis; most notably, the use of UPLC-MS/MS demonstrated that samples can be reliably screened with significantly reduced analysis times.
Keywords: UPLC; Tandem mass spectrometry; Diuretics; Beta-blockers; Stimulants; Urine; Doping;

S-adenosyl methionine (SAM) is an important methyl group donor that is formed from methionine. S-adenosyl homocysteine (SAH) is formed after demethylation of SAM and represents a potent inhibitor of many methyltransferases. We developed an improved stable-isotope dilution ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method for the simultaneous quantification of SAM and SAH in biological samples. The method comprises a phenylboronic acid-containing solid-phase extraction procedure, serving for binding and clean-up of SAM and SAH. After extraction, samples were separated and detected using either a HPLC SymmetryShield RP18 or an Acquity UPLC BEH C18 column with a HPLC–MS/MS or an UPLC–MS/MS system. The best results were obtained by Acquity UPLC BEH C18 column. In plasma samples, the estimated intraassay coefficients of variation (CVs) for SAM and SAH were 3.3% and 3.9%, respectively, the interassay CVs were 10.1% for SAM and 8.3% for SAH. Mean recovery of SAM and SAH at two different concentrations was 100.0% for SAM and 101.7% for SAH. The quantification limits were 0.5 and 0.7 nmol/L for SAM and SAH, respectively. In 31 plasma samples, the mean concentrations (SD) were 85.5 (11.1) nmol/L for SAM and 13.3 (5.0) nmol/L for SAH with a SAM/SAH ratio of 7.0 (1.8). The new UPLC–MS/MS method showed very high sensitivity and selectivity for SAM and SAH, low CVs and fast sample preparation (40 samples in 60 min) and analysis time (3 min). This new assay can be used for large-scale clinical studies.
Keywords: S-adenosyl methionine; S-adenosyl homocysteine; Homocysteine; Tandem mass spectrometry;

This paper described the development and validation of a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the quantitative determination of 6-benzylthioinosine (6BT), a novel myeloid leukemia differentiation-inducing agent, in mouse and human plasma. In this method, 2-amino-6-benzylthioinosine (2A6BT) was used as internal standard and ethyl acetate was used as organic solvent for the extraction of 6BT and internal standard from plasma samples. The extracted samples were separated on YMC ODS-AQ® column (2.0 mm × 50 mm), and the eluates from the column were monitored by the positive-electrospray-ionization tandem mass spectrometer (ESI+-MS/MS). Quantification of 6BT by internal calibration with 2A6BT was carried out using multiple-reaction-monitoring (MRM) mode. This method had a linear calibration range of 3.00–1.00 × 103  ng/mL with correlation coefficients of 1.00 and 0.999 in mouse and human plasma. The lower limit of quantification (LLOQ) was 3.00 ng/mL in either mouse or human plasma. The method had recovery of 6BT of 82–87% from mouse plasma and 90–98% from human plasma. Both accuracy (as percent error) and precision (as coefficient of variation) of the method were ≤±15% within the calibration range. This method had been successfully applied to the pharmacokinetic study of 6BT in male C57 mice with intraperitoneal (i.p.) injection at the dose of 1 mg 6BT per kilogram mouse.
Keywords: 6-Benzylthioinosine; LC–MS/MS; Myeloid leukemia differentiation-inducing agent; Mouse and human plasma;

A comparison of HPLC/APCI-MS and MALDI-MS for characterising triacylglycerols in insects: Species-specific composition of lipids in the fat bodies of bumblebee males by Edita Kofroňová; Josef Cvačka; Vladimír Vrkoslav; Robert Hanus; Pavel Jiroš; Jiří Kindl; Oldřich Hovorka; Irena Valterová (3878-3884).
Two mass spectrometric methods for analysing triacylglycerols (HPLC/APCI-MS and MALDI-MS) were used and compared in terms of the relevance of the data for further biostatistical evaluation. While MALDI-MS is simpler and significantly faster, the time-consuming and labour-intensive HPLC/APCI-MS provides more complete information about the lipid components. However, both methods provide well-comparable results concerning the grouping of specimens belonging to different species when evaluated with multivariate exploratory approaches. The compositions of triacylglycerols in the fat bodies of males in 11 bumblebee species (Bombus terrestris, B. lucorum, B. lapidarius, B. pratorum, B. sylvarum, B. ruderatus, B. pomorum, B. subterraneus, B. campestris, B. bohemicus, and B. rupestris) were found to be species-specific.
Keywords: Insect lipids; MALDI; APCI; Mass spectrometry; Multivariate exploratory methods; Honeybee;

Global histone modifications and their putative relevance to short and long term cellular programming have drawn substantial interest in the study of chromatin. Here we describe the use of reverse-phase liquid chromatography coupled to Linear Ion Trap-Fourier Transform Mass Spectrometry (RPLC–LTQ-FTMS) to quickly profile post-translationally modified isoforms and variants for core histone proteins from as few as 5 × 104 cells at isotopic resolution. Such LC–MS profiling greatly facilitated the detection of histones from HeLa S3 or 293T cells experiencing shRNA- or siRNA-knockdown of histone deacetylase (HDAC) 1, 2, 3 or 1 and 2 together. In no case was significant global histone hyperacetylation relative to control cells observed, suggesting possible compensation of deacetylation activity by partially redundant enzymes in the 18-member HDAC family. This contrasts sharply with yeast where genetic deletion of HDAC rpd3 causes massive hyperacetylation. Treatment of cells with TSA and class I selective HDAC inhibitors had similar ability to induce global histone hyperactylation, though to different extents in HeLa S3 vs. 293T cells.
Keywords: Acetylation; Histone; HDACs; LC–FTMS;

Quantification of coproporphyrin isomers I and III in urine by HPLC and determination of their ratio for investigations of Multidrug Resistance Protein 2 (MRP2) function in humans by Renaud Respaud; Isabelle Benz-de Bretagne; Helene Blasco; Jean Sébastien Hulot; Philippe Lechat; Chantal Le Guellec (3893-3898).
We describe here the development of a high-performance liquid chromatography (HPLC) method for quantitative determination of the ratio of isomers I and III of urinary coproporphyrin [the UCP I/(I + III) ratio], which is used for the diagnosis of Dubin–Johnson syndrome (DJS). This technique could also be used for research applications, such as investigations of the function of Multidrug Resistance Protein 2 (MRP2) in humans. Chromatographic separation was achieved on a reverse-phase C18 Symmetry® column (5 μm; 4.8 mm × 250 mm), using a mobile phase consisting of a mixture of acetonitrile and acetate buffer (0.015 M, pH 4), with fluorescence detection based on excitation at 365 nm and emission at 624 nm. The method was validated over a concentration range of 10–400 nmol/l for UCP I and 30–560 nmol/l for UCP III, yielding calibration curves with correlation coefficients greater than 0.998. The lower limit of quantification (LLOQ) was 7 nmol/l for UCP I and 10 nmol/l for UCP III. Inter- and intra-day precision (CV < 5%) and accuracy (95–99%) complied with ICH guidelines. We also demonstrated that samples could be stored for 3 days at +4 °C and for 12 months at −20 °C with no change in UCP ratio (CV < 5%), providing a basis for storage recommendations for future clinical studies based on this analysis. Our method is simple, rapid and universal and is suitable for quantitative determinations of each isomer and their ratio for routine and research purposes.
Keywords: Coproporphyrins; High-performance liquid chromatography; ATP-binding cassette transporters; Multidrug Resistance-associated Protein 2; Biological markers;

Liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry method for the quantification of pregabalin in human plasma by Ramakrishna Nirogi; Vishwottam Kandikere; Koteshwara Mudigonda; Prashanth Komarneni; Raghupathi Aleti (3899-3906).
A sensitive high-performance liquid chromatography positive ion atmospheric pressure chemical ionization tandem mass spectrometry method was developed and validated for the quantification of pregabalin in human plasma. Following liquid–liquid extraction, the analyte was separated using an isocratic mobile phase on a reverse-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 160–142 for pregabalin and m/z 482–258 for the internal standard. The assay exhibited a linear dynamic range of 1–10,000 ng/mL for pregabalin in human plasma. The lower limit of quantification was 1 ng/mL with a relative standard deviation of less than 11.4%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 4.0 min for each sample made it possible to analyze more than 300 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic studies.
Keywords: Pregabalin; Liquid chromatography–tandem mass spectrometry; Atmospheric pressure chemical ionization; Human plasma; Pharmacokinetic study;

A quantitative HPLC method with fluorescence detection has been developed for the simultaneous determination of four anthracyclines (doxorubicin, epirubicin, daunorubicin and idarubicin) and their respective 13-S-dihydro metabolites (doxorubicinol, epirubicinol, daunorubicinol and idarubicinol) in plasma and saliva, using epidaunorubicin as internal standard. A progressive optimization matrix led to a two-step extraction based on a protein precipitation with ethanol followed by a liquid–liquid extraction with dichloromethane after pH adjustment to 8.5. The chromatographic separation was performed in 14 min on a C18 column, applying gradient elution with a mixture of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. The analytes were detected and quantified at an excitation and emission wavelength of 480 and 555 nm, respectively. Limit of detection (LOD) and lower limit of quantification (LLOQ) were 0.3 or 0.75, and 1 or 2.5 ng/mL, respectively. Linearity by means of weighted (1/x) regression was obtained from the LLOQ up to 1000 or 2500 ng/mL for the parent drugs and up to 400 or 1000 ng/mL for the metabolites. Intra-assay and inter-assay relative standard deviation values were all less than 14% at low, medium and high levels, and below 17% at the LLOQ. Accuracy ranged between 91 and 113% at low, medium and high concentrations and between 83 and 118% at the LLOQ. Absolute recoveries were between 78 and 88% in plasma, and between 70 and 79% in saliva, respectively. Autosampler, benchtop, freeze–thaw and long-term stability samples fulfilled acceptance criteria. This selective method was applied successfully to the analysis of plasma and saliva samples from patients administered epirubicin intravenously.
Keywords: Anthracyclines; Doxorubicin; Epirubicin; Daunorubicin; Idarubicin; Doxorubicinol; Epirubicinol; Daunorubicinol; Idarubicinol; Plasma; Saliva; Liquid–liquid extraction (LLE); Liquid chromatography; Fluorescence detection; Method validation;

Analysis of mycophenolic acid in dried blood spots using reversed phase high performance liquid chromatography by A.J. Wilhelm; J.C.G. den Burger; A. Chahbouni; R.M. Vos; A. Sinjewel (3916-3919).
At our laboratory a reversed phase high performance liquid chromatography assay was developed for analysis of mycophenolic acid in dried blood spot samples. The assay was validated in the range of 0.74–23.4 mg/L and proved to be accurate and precise. The developed sample pretreatment procedure was consistent and has a recovery of 95.2% for mycophenolic acid. Hematocrit showed to have influence on the physics of the blood spots and thus on the concentration of mycophenolic acid, therefore standard and control samples should be made with a standardized hematocrit.
Keywords: Mycophenolic acid; Dried blood spots; Hematocrit; Fingerprick;

Rapid quantification of bile acids and their conjugates in serum by liquid chromatography–tandem mass spectrometry by Max Scherer; Carsten Gnewuch; Gerd Schmitz; Gerhard Liebisch (3920-3925).
Beside their role as lipid solubilizers, bile acids (BAs) are increasingly appreciated as signaling factors. As ligands of G-protein coupled receptors and nuclear hormone receptors BAs control their own metabolism and act on lipid and energy metabolism. To study BA function in detail, it is necessary to use methods for their quantification covering the structural diversity of this group. Here we present a simple, sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the analysis of bile acid profiles in human plasma/serum. Protein precipitation was performed in the presence of stable-isotope labeled internal standards. In contrast to previous LC–MS/MS methods, we used a reversed-phase C18 column with 1.8 μm particles and a gradient elution at basic pH. This allows base line separation of 18 bile acid species (free and conjugated) within 6.5 min run time and a high sensitivity in negative ion mode with limits of detection below 10 nmol/L. Quantification was achieved by standard addition and calibration lines were linear in the tested range up to 28 μmol/L. Validation was performed according to FDA guidelines and overall imprecision was below 11% CV for all species. The developed LC–MS/MS method for bile acid quantification is characterized by simple sample preparation, baseline separation of isobaric species, a short analysis time and provides a valuable tool for both, routine diagnostics and the evaluation of BAs as diagnostic biomarkers in large clinical studies.
Keywords: Liquid chromatography tandem mass spectrometry (LC–MS/MS); Bile acid; Deuterated internal standard; Method validation; Quantification;

The determination of phenylalanine and tyrosine is presently the most reliable direct approach to the diagnosis of phenylketonuria. An HPLC method for the simultaneous measurement of phenylalanine and tyrosine in samples of dried blood spots and plasma has been developed and evaluated. We have used an inherent fluorescence of both phenylalanine and tyrosine. For the separation, a reverse-phase column LiChroCart 125-4, Purospher RP-18e, 5 μm, was used. The mixture of ethanol and deionized water (5:95, v/v) was used as a mobile phase. Analytical performance of this method is satisfactory for both phenylalanine and tyrosine: the intra-assay and inter-assay coefficients of variation were below 10%. Quantitative recoveries from spiked plasma and blood samples were between 92.0 and 102.9%. The limit of detection was 10.0 and 5.0 μmol/L, respectively. The preliminary reference ranges of phenylalanine and tyrosine in a group of newborns are 69.3 ± 13.1 and 42.7 ± 12.9 μmol/L, in a group of blood donors are 68.4 ± 9.9 and 52.1 ± 10.9 μmol/L. The presented method is inexpensive and suitable for diagnosis of phenylketonuria.
Keywords: Dried blood spots; HPLC; Fluorescence detection; N-Methylphenylalanine; Phenylalanine; Phenylketonuria; Tyrosine;