Liquid chromatography setup-dependent artefactual methionine oxidation of peptides: The importance of an adapted quality control process.

In both biologics quality control experiments and protein post-translational modification studies, the analytical system used is not supposed to bring any artefactual modifications which could impair the results. In this work, we investigated oxidation of methionine-containing peptides during reversed-phase (RP) chromatographic separation. We first used a synthetic methionine-containing peptide to evaluate this artefactual phenomenon and then considered more complex samples (i.e., plasma and HeLa protein digests). The methionine oxidation levels of the peptides were systematically assessed and compared for the long-term use of the analytical column, the sample trapping time, the gradient length, the sample load and the nature of the stationary phase (HSS T3 from Waters, YMC Triart C18 from YMC Europe GmbH and BEH130 C18 from Waters). In addition to the oxidation of methionine in solution, we observed on the HSS T3 and the BEH130 stationary phases an additional broad peak corresponding to an on-column oxidized species. Considering the HSS T3 phase, our results highlight that the on-column oxidation level significantly increases with the age of the analytical column and the gradient length and reaches 56 % when a 1-year-old column set is used with a 180 min-long LC method. These levels go to 0 % and 18 % for the YMC Triart C18 and the BEH130 C18 phases respectively. Interestingly, the on-column oxidation proportion decreases as the injected sample load increases suggesting the presence of a discrete number of oxidation sites within the stationary phase of the analytical column. Those findings observed in different laboratories using distinct set of columns, albeit to varying degrees, strengthen the need for a standard of methionine-containing peptide that could be used as a quality control to appraise the status of the liquid chromatographic columns.

A validated RP-HPLC method for the determination of piperidone analogue of curcumin.

Curcumin (Diferuloylmethane) is a natural product extracted from the root of Curcuma longa. 5-Bis (4-hydroxy-3-methoxybenzylidene)-N-methyl-4-piperidone, the piperidone analogue of curcumin (PAC), was one of the analogues that, demonstrated potential anticancer effects against breast and colon cancers compared with native curcumin. A simple, accurate, and rapid isocratic reverse phase high performance liquid chromatography (HPLC) analytical method utilizing UV detection was developed and validated for the determination of PAC utilizing C18 column with run time was 7 min. Chromatogram showed a peak of PAC at retention time of 5.8±0.92 min. The method was validated for linearity, accuracy, precision, limit of detection, limit of quantitation and robustness. Linear relationship (r > 0.99) was observed between AUP of PAC and the corresponding concentrations over 100-10000µg/mL. The LOQ of this assay was 3.9ng/mL with a corresponding relative standard deviation of 4.8 and 4.0%. The LOD was 13.1ng/mL at a signal-to-noise ratio of >3.

Qualitative evaluation of high pH mass spectrometry-compatible reversed phase liquid chromatography for altered selectivity in separations of intact proteins.

Intact proteins are increasingly being recognized as potential biomarkers and biotherapeutic agents for cancer and other serious diseases. Low pH reversed phase plays an important role in both single and multidimensional protein separations for resolving complex protein samples prior to mass spectrometric detection. In this work, we evaluated the use of high pH reversed phase liquid chromatography as an alternative chromatographic separation to gain different selectivity while maintaining the high resolving power and MS compatibility of reversed phase separations. The altered selectivity gained by high pH reversed phase liquid chromatography can further help to separate unresolved protein peaks or to increase peak capacity and resolving power of a multidimensional setup for complex biological samples. Hence, we evaluated the use of different MS-friendly buffers, ion pairing reagents, and stationary phases (silica- and polymer-based) at alkaline pH for intact protein separations. The best chromatographic separation, with complementary selectivity to low pH reversed phase, was achieved using triethylammonium bicarbonate at pH 10 and hybrid silica particles.

Development and validation of simple step protein precipitation UHPLC-MS/MS methods for quantitation of temozolomide in cancer patient plasma samples.

Temozolomide (TEMODAL™) (TMZ) is an antineoplastic agent that is primarily used for the treatment of glioblastoma and anaplastic gliomas, two aggressive forms of brain cancer. Due to the poor prognosis of brain tumour patients, there is an increasing body of research into improving the stability and delivery of TMZ past the blood brain barrier using carrier molecules. These require accurate determination of TMZ levels for biodistribution and pharmacokinetic evaluation. Unfortunately, current methodologies for the determination of TMZ in human plasma suffer from low reproducibility, recovery, sensitivity or cost ineffective procedures associated with extensive sample cleaning. To surpass these disadvantages, we developed two bioanalytical methods with high sensitivity and excellent recovery for the determination of TMZ in human plasma at minimum cost. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used and both methods were validated under US Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) guidelines. The two methods had minor differences in the sample pre-treatment and each method was developed and applied in separate laboratories. Theophylline was selected as internal standard (IS). Calibration curves were linear over the range of 10-500 ng/mL with extraction recovery ranging from 77.3 to 97.3% while all validation parameters met the acceptance criteria and proved the methods' reliability. The validated methods were successfully applied to plasma samples donated from cancer patient following treatment with temozolomide.

A new, sensitive and versatile assay for quantitative determination of α-fluoro-ß-alanine (AFBA) in human urine by using the reversed-phase ultrahigh performance-tandem mass spectrometry (rp-UHPLC-MS/MS) system.

A method for the quantitation of α-fluoro-ß-alanine (AFBA), the main metabolite of capecitabine (Cape) and 5-fluoruracil (5-FU), is described. Among antineoplastic drugs (ADs), 5-FU and Cape (the new oral prodrug) are the most commonly applied drugs in cancer therapy. The main objective of this study was to develop a reliable method that would be easy to run on a reversed-phase UHPLC system coupled to tandem mass spectrometry. AFBA was derivatized with Sanger's reagent to ensure complete yield of a stable 2,4 dinitrophenil-α-fluoro-ß-alanine derivative. This method was based on the use of a mixed-mode anion exchange solid phase extraction enabling urinary extracts to be clear of endogenous interferences affecting quantitative results. The assay was validated in human urine according to FDA criteria with the use of a labeled internal standard (ß-alanine-d4) to minimize experimental error. Good accuracy and precision were demonstrated by determining spiked urine QC samples in four consecutive days. The recovery of AFBA was between 70.0 and 82.6%, with a matrix effect that was 12.8%-18.5%. The lower limit of quantitation (LOQ) was 0.5 ng/mL with a coefficient of variation of 5.3%. This assay was successfully applied to determine the levels of this metabolite in a large number of urine samples taken from personnel who were occupationally exposed to ADs.

Liquid chromatography-tandem mass spectrometric assay for the quantitation of the novel radiation protective agent and radiation mitigator JP4-039 in murine plasma.

JP4-039 radio-protects prior to, and radio-mitigates after ionizing radiation by neutralizing reactive oxygen species. We developed and validated an LC-MS/MS assay for the quantitation of JP4-039 in murine plasma. Methanol protein precipitation of 50µL plasma was followed by isocratic reverse phase chromatography for a 6min run time, and electrospray positive mode ionization mass spectrometric detection. The plasma assay was linear from 1 to 1000ng/mL with appropriate accuracy (97.1-107.6%) and precision (3.7-12.5%CV), and fulfilled FDA guidance criteria. Recovery was 77.2-136.1% with moderate ionization enhancement (10.9-39.5%). Plasma freeze-thaw stability (98.8-104.2%), stability for 13.5 months at -80°C (93.1-105.6%), and stability for 4h at room temperature (94.2-97.6%) were all acceptable. Limited cross-validation to tissue homogenates suggested that these could also be analyzed for JP4-039 accurately. This assay has been directly applied to determine the pharmacokinetics of JP4-039 in C57BL/6 male mice after IV administration of 20mg/kg JP4-039 and will be extended to other studies of this agent.

Development and validation of a bioanalytical method for quantification of LNA-i-miR-221, a 13-mer oligonucleotide, in rat plasma using LC-MS/MS.

LNA-i-miR-221, a 13-mer oligonucleotide, is a new miR-221 inhibitor that could be used as a novel drug for multiple myeloma. Herein, an ion-pair reversed phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the quantification of LNA-i-miR-221 in rat plasma. Plasma samples were prepared with an initial phenol/chloroform/isoamyl alcohol liquid-liquid extraction followed by a solid phase extraction. Chromatographic separation was performed with a gradient system on a HALO C18 column using hexafluoro-2-propanol/triethylamine buffer and methanol as mobile phase at a flow rate of 0.4 mL/min. Under these conditions LNA-i-miR-221 and the analogue internal standard are co-eluted at 1.2 min. The detection was carried out in multiple reaction monitoring (MRM) mode using a negative electrospray ionization (ESI) interface. The assay showed a good linearity within the calibration range 10-10000 ng/mL. The precision, accuracy, and recovery values were found to be <15% (<20% at LLOQ), 100 ±â€¯15%, and 97.6-103.7%, respectively. This method was successfully applied to measure the concentrations of LNA-i-miR-221 in plasma samples following the intravenous administration during a 4-week toxicity study in rats.

Stability assessment of antibody-drug conjugate Trastuzumab emtansine in comparison to parent monoclonal antibody using orthogonal testing protocol.

Antibody-drug conjugates (ADC) represent an emerging, novel class of biopharmaceuticals. The heterogeneity originating from the sophisticated structure requires orthogonal analytical techniques for quality and stability assessment of ADC to ensure safety and efficacy. In this study, the stability of Trastuzumab (recombinant humanized IgG1 mAb, targeting HER2 receptor) and its ADC with DM1 (anti-tubulin anticancer drug), Trastuzumab emtansine (T-DM1) were studied. SE-HPLC was used to monitor formation of aggregates and/or fragments of the monoclonal antibodies (mAb). Correlation with the results of reducing and non-reducing sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE) and dynamic light scattering (DLS) were performed to interpret the obtained results. RP-HPLC was used for assessment of the stability of DM1 in ADC while spectrophotometry was employed to determine drug antibody ratio (DAR) . The studied drugs were subjected to several stress conditions including pH, temperature, mechanical agitation and repeated freeze and thaw to generate possible degradation products and ensure suitability of the assay protocol. The degradation pattern and extent were demonstrated under the indicated stress conditions. The correlation between the results of SE-HPLC and those of SDS-PAGE and DLS ensured the validity of the orthogonal assay protocol and indicated aggregates that were not detected using SE-HPLC. Results showed clearly that T-DM1 is relatively less stable than its parent mAb. This was attributed to the presence of the drug-linker part that is attached to the mAb. RP-HPLC showed that the cytotoxic drug moiety is liable for degradation under the studied conditions resulting in alteration of DAR as well as formation of degradation products. This confirmed the need for more robust coupling chemistries for production of safe and effective ADC and highlighted the importance of orthogonal testing protocols for quality assessment. The assay protocol should be applicable for quality and stability assessment of various ADC.

Development and validation of a simple and robust HPLC method with UV detection for quantification of the hepatitis C virus inhibitor daclatasvir in human plasma.

Daclatasvir is an inhibitor of hepatitis C virus NS5A protein that is used for the therapy of chronic hepatitis. So far, published methods for analysis of daclatasvir in plasma are exclusively based on mass spectrometry, which is not always available in standard clinical laboratories. Thus, we wished to develop and validate a simple, but still reliable and sensitive high-performance liquid chromatography (HPLC) assay with UV detection for the quantification of daclatasvir, feasible for a wide-spread clinical routine use. The method consisted of solid-phase extraction of daclatasvir using Waters Oasis HLB 1cc cartridges, reversed-phase liquid chromatography with a Waters XTerra RP18 (150mm×4.6mm, 3.5µm) column and a mobile phase of ammonium acetate buffer (pH 5.0, 10mM) and acetonitrile (56:44, v/v), and UV detection at 318nm. This assay proved to be sensitive (lower limit of quantification of 0.05µg/mL), linear (correlation coefficients ≥0.997), specific (no interference with various potentially co-administrated drugs), reproducible (both intra-day and inter-day coefficients of variation ≤8.9%), and accurate (deviations ranged from -2.2 to 8.0% and from -6.5 to 9.2% for intra-day and inter-day assays, respectively). The method was applied to therapeutic monitoring of patients undergoing daclatasvir therapy for hepatitis C and showed to be reliable and robust. Thus, this method provides a simple, sensitive, precise, and reproducible assay for dosing daclatasvir that can be readily adaptable to routine use by clinical laboratories with standard equipment. In addition, the stability of daclatasvir in plasma was evaluated under various conditions, including after the heating procedure required for inactivation of infectious viruses and in different light exposure conditions. These studies evidenced photo-instability of the compound under sunlight exposure over time. Thus, blood sampling and the whole handling procedure have to be performed quickly and with minimal light exposure.

REVERSE PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR DETERMINATION OF 5-FLUOROURACIL IN RABBIT PLASMA.

A simple, efficient, accurate and selective HPLC method has been developed and validated successfully for the estimation of 5-fluorouracil in rabbit plasma. The drug was eluted by using Supelco C18 column (1.5 cm x 4.6 mm, 5 µm) with a mobile phase consisting of methanol and water (20: 80, v/v) by adjusting pH at 3.2, using perchloric acid solution. The retention time was found to be 4.107 with a flow rate of I mL/min. Multiple validation parameters evaluated with high accuracy indicating that the current method sufficiently qualifies the recommended criteria. Regression value obtained from linearity curve was R² = 0.999 and percentage recovery obtained was ranged from 96.6 to 102.5%. A fine response in short run time with perfect resolution made the method highly suitable for pharmacokinetic studies.

Efficient Microscale Basic Reverse Phase Peptide Fractionation for Global and Targeted Proteomics.

Analysis of small biological samples would benefit from an efficient microscale fractionation strategy that minimizes sample handling, transfer steps, and accompanying losses. Here we describe a microscale basic reverse phase liquid chromatographic (bRPLC) fractionation method that offers high reproducibility and efficiency for peptide mixtures from small (5-20 µg) samples. We applied our platform to detect differentially expressed proteins from lung tumor cell lines that are sensitive (11-18) and resistant (11-18R) to the tyrosine kinase inhibitor erlotinib. Label-free analyses of 5-20 µg samples yielded identifications of approximately 3,200 to 4,000 proteins with coefficients of variation of 1.9-8.9% in replicate analyses. iTRAQ analyses produced similar protein inventories. Label-free and iTRAQ analyses displayed high concordance in identifications of proteins differentially expressed in 11-18 and 11-18R cells. Micro-bRPLC fractionation of cell proteomes increased sensitivity by an average of 4.5-fold in targeted quantitation using parallel reaction monitoring for three representative receptor tyrosine kinases (EGFR, PDGFRA, and BMX), which are present at low abundance in 11-18 and 11-18R cells. These data illustrate the broad utility of micro-bRPLC fractionation for global and targeted proteomic analyses. Data are available through Proteome eXchange Accession PXD003604.

Exploitation of the size-exclusion effect of reversed-phase high performance liquid chromatography for the direct analysis of diethylene triamine pentaacetic acid in therapeutic monoclonal antibody formulations.

Monoclonal antibodies (mAb) are being widely studied for the treatment of cancers and other diseases. The mAb is typically in a solution formulation and administered as an intravenous infusion. Ready-to-use solutions are favored for their clinical convenience but they can potentially suffer from a shorter shelf life due to accelerated rates of some forms of degradation such as oxidation, relative to lyophilized formulations. To improve stability, the chelating agent diethylene triamine pentaacetic acid (DTPA) is often used at very low concentrations in biologics formulations to prevent oxidation induced by metal ions. Because of its low concentration and susceptibility to changes in concentration during stability study or processing, the measurement of DTPA levels during formulation and process development is critical. In response to this need we developed a platform reversed-phase HPLC method that allows for the rapid and direct determination of DPTA concentrations which does not require the prior removal of mAbs in formulation samples. The method exploits the "size exclusion effect" of C18 columns with narrow pore sizes (90-120Å) to elute large mAb at the void volume, enabling direct injections of mAb samples for quantitation of DTPA. The method was found to be suitable for the analysis of DTPA in the range of 2-20µg/mL across multiple drug formulations containing different therapeutic mAb and antibody drug conjugates. The method was successfully validated for specificity, precision, accuracy, linearity, and robustness.

Development and Validation of a High Pressure Liquid Chromatography-UV Method for the Determination of Treosulfan and Its Epoxy Metabolites in Human Plasma and Its Application in Pharmacokinetic Studies.

Treosulfan (l-threitol-1,4-di-methanesulfonate) is a prodrug of a bifunctional alkylating agent that is being used increasingly in pediatric bone marrow transplantation regimens. The activation pathway is a complex reaction, which consists of two consecutive reactions leading to epoxybutane derivatives which are responsible for DNA alkylation. A simple, sensitive high performance liquid chromatography method for the determination of the sum of treosulfan and its epoxy metabolites by UV detection after derivatization with sodium diethyldithiocarbamate in human plasma was developed and validated. Plasma samples containing treosulfan and epoxy metabolites were converted into thiocarbamate derivative with 10% sodium diethyldithiocarbamate. Dinitrobiphenyl was used as an internal standard. The analysis was carried out using a reversed phase C18 column with a mobile phase consisting of methanol-water (65:35, v/v) at a flow rate of 1 mL/min. The eluent was monitored at 254 nm. The standard calibration curve was established between 2.5 and 50 µg/mL, with a correlation coefficient of 0.9987. Intra- and interday precision and accuracy of the method was <8% and met the analytical criteria. Pharmacokinetic parameters were determined in six children who received intravenous treosulfan (dose range 12-24 g/m(2)) in combination with fludarabine prior to blood or marrow transplantation.

Purine metabolite and energy charge analysis of Trypanosoma brucei cells in different growth phases using an optimized ion-pair RP-HPLC/UV for the quantification of adenine and guanine pools.

Human African Trypanosomiasis (HAT) is caused by the protozoan parasite Trypanosoma brucei. Although trypanosomes are well-studied model organisms, only little is known about their adenine and guanine nucleotide pools. Besides being building blocks of RNA and DNA, these nucleotides are also important modulators of diverse biochemical cellular processes. Adenine nucleotides also play an important role in the regulation of metabolic energy. The energetic state of cells is evaluated by the energy charge which gives information about how much energy is available in form of high energy phosphate bonds of adenine nucleotides. A sensitive and reproducible ion-pair RP-HPLC/UV method was developed and optimized, allowing the quantification of guanine and adenine nucleosides/nucleotides in T. brucei. With this method, the purine levels and their respective ratios were investigated in trypanosomes during logarithmic, stationary and senescent growth phases. Results of this study showed that all adenine and guanine purines under investigation were in the low mM range. The energy charge was found to decrease from logarithmic to static and to senescent phase whereas AMP/ATP, ADP/ATP and GDP/GTP ratios increased in the same order. In addition, the AMP/ATP ratio varied as the square of the ADP/ATP ratio, indicating AMP to be the key energy sensor molecule in trypanosomes.

Optimization and validation of a reversed-phase high performance liquid chromatography method for the measurement of bovine liver methylmalonyl-coenzyme a mutase activity.

BACKGROUND: Methylmalonyl-CoA mutase (MCM) is an adenosylcobalamin-dependent enzyme that catalyses the interconversion of (2R)-methylmalonyl-CoA to succinyl-CoA. In humans, a deficit in activity of MCM, due to an impairment of intracellular formation of adenosylcobalamin and methylcobalamin results in a wide spectrum of clinical manifestations ranging from moderate to fatal. Consequently, MCM is the subject of abundant literature. However, there is a lack of consensus on the reliable method to monitor its activity. This metabolic pathway is highly solicited in ruminants because it is essential for the utilization of propionate formed during ruminal fermentation. In lactating dairy cows, propionate is the major substrate for glucose formation. In present study, a reversed-phase high performance liquid chromatography (RP-HPLC) was optimized and validated to evaluate MCM activity in bovine liver. The major aim of the study was to describe the conditions to optimize reproducibility of the method and to determine stability of the enzyme and its product during storage and processing of samples. RESULTS: Specificity of the method was good, as there was no interfering peak from liver extract at the retention times corresponding to methylmalonyl-CoA or succinyl-CoA. Repeatability of the method was improved as compared to previous RP-HPLC published data. Using 66 µg of protein, intra-assay coefficient of variation (CV) of specific activities, ranged from 0.90 to 8.05% and the CV inter-day was 7.40%. Storage and processing conditions (frozen homogenate of fresh tissue vs. fresh homogenate of tissue snapped in liquid nitrogen) did not alter the enzyme activity. The analyte was also stable in liver crude extract for three frozen/thawed cycles when stored at -20°C and thawed to room temperature. CONCLUSIONS: The improved method provides a way for studying the effects of stages of lactation, diet composition, and physiology in cattle on MCM activity over long periods of time, such as a complete lactation period. Interestingly, this sensitive and accurate method could benefit the study of the cobalamin status in experimental studies and clinical cases.

Mixed-bed ion exchange chromatography employing a salt-free pH gradient for improved sensitivity and compatibility in MudPIT.

In proteomics, comprehensive analysis of peptides mixtures necessitates multiple dimensions of separation prior to mass spectrometry analysis to reduce sample complexity and increase the dynamic range of analysis. The main goal of this work was to improve the performance of (online) multidimensional protein identification technology (MudPIT) in terms of sensitivity, compatibility and recovery. The method employs weak anion and strong cation mixed-bed ion exchange chromatography (ACE) in the first separation dimension and reversed phase chromatography (RP) in the second separation dimension (Motoyama et.al. Anal. Chem 2007, 79, 3623-34.). We demonstrated that the chromatographic behavior of peptides in ACE chromatography depends on both the WAX/SCX mixing ratio as the ionic strength of the mobile phase system. This property allowed us to replace the conventional salt gradient by a (discontinuous) salt-free, pH gradient. First dimensional separation of peptides was accomplished with mixtures of aqueous formic acid and dimethylsulfoxide with increasing concentrations. The overall performance of this mobile phase system was found comparable to ammonium acetate buffers in application to ACE chromatography, but clearly outperformed strong cation exchange for use in first dimensional peptide separation. The dramatically improved compatibility between (salt-free) ion exchange chromatography and reversed phase chromatography-mass spectrometry allowed us to downscale the dimensions of the RP analytical column down to 25 µm i.d. for an additional 2- to 3-fold improvement in performance compared to current technology. The achieved levels of sensitivity, orthogonality, and compatibility demonstrates the potential of salt-free ACE MudPIT for the ultrasensitive, multidimensional analysis of very modest amounts of sample material.

Targeted mass spectrometry-based metabolomic profiling through multiple reaction monitoring of liver and other biological matrices.

In a systemic viewpoint, relevant biological information on living systems can be grasped from the study of small, albeit pivotal molecules which constitute the fundamental bricks of metabolic pathways. This holds true for liver which plays, among its unique functions, a key role in metabolism. The nonbiased analysis of all this small-molecule complement in its entirety is known as metabolomics. However, no practical approach currently exists to investigate all metabolic species simultaneously without including a technical bias towards acidic or basic compounds, especially when performing mass spectrometry-based investigations. Technical aspects of rapid resolution reversed phase HPLC online with mass spectrometry are hereby described. Such an approach allows to discriminate and quantify a wide array of metabolites with extreme specificity and sensitivity, thus enabling to perform complex investigations even on extremely low quantities of biological material. The advantages also include the possibility to perform targeted investigations on a single (or a handful of) metabolite(s) simoultaneously through single (multiple) reaction monitoring, which further improves the dynamic range of concentrations to be monitored.Such an approach has already proven to represent a valid tool in the direct (on the liver) or indirect (on human red blood cell metabolism which is hereby presented as a representative model, but also on blood plasma or other biological fluids) assessment of metabolic poise modulation and pharmacokinetics for drug development.

Development and validation of LC-MS/MS method for the detection and quantification of CpG oligonucleotides 107 (CpG ODN107) and its metabolites in mice plasma.

CpG oligodeoxynucleotide 107 (CpG ODN107) could be used as a novel radiosensitizer for glioma. Herein, a novel and sensitive reversed-phase HPLC coupled with electrospray triple quadrupole mass spectrometry (LC-MS/MS) following a one-step C18 solid-phase extraction (SPE) for biological matrix removal was developed and fully validated for the determination of CpG ODN107 and its metabolites such as 5'N-1, 3'N-1, 3'N-2, and 3'N-3 in mouse plasma. The analytes were separated on an Extend-C18 analytical column (150 mm × 2.1 mm, 3.5 µm) using an eluent of acetonitrile-0.05% aqueous NH(3) (20:80, v/v) and detected by electrospray ionization (ESI) mass spectrometry in the negative multiple reaction monitoring mode (MRM). The assay was specific, and it showed a good linearity with a determination coefficient (r(2)) that was greater than or equal to 0.998 for CpG ODN107 and its metabolites in the biological matrices. The precision, accuracy, and relative recovery values were found to be <15%, ±15%, and 95-105%, respectively. This method was successfully applied to measure the concentrations of CpG ODN107 and its metabolites in the plasma following the intravenous administration of 15.0 mg/kg of CpG ODN107 in mice; therefore, the method was suitable for preclinical pharmacokinetic studies on CpG ODN107 and its metabolites.

Development of a LC-MS method for quantification of FK-3000 and its application to in vivo pharmacokinetic study in drug development.

FK-3000 can inhibit proliferation of carcinomas and arrest the growth of carcinoma cells through cytotoxic (apoptosis induction) and cytostatic (cell cycle arrest) effects. A rapid and sensitive assay was developed and validated using liquid chromatography-mass spectrometry (LC-MS) for FK-3000 in rat plasma. FK-3000 was extracted with ethyl acetate from rat plasma samples, and the residue containing the FK-3000 was dried in a gentle stream of nitrogen and reconstituted with acetonitrile. The FK-3000 was quantified using high-performance liquid chromatography (HPLC; Waters Alliance 2695) with a reversed phase Gemini column (3 mm × 150 mm, 5 µm; Phenomenex, USA) and a Waters Micromass ZQ detector. FK-3000 and phenazine, an internal standard (IS), were analyzed by selected ion monitoring (SIM) at m/z transitions of 418.45 and 256, respectively. A lower limit of quantification (LLOQ) of 10 ng/mL was observed, with a linear dynamic range from 10 to 10,000 ng/mL (R>0.999). The accuracy, precision, recovery, matrix effects, and stability of the assay were deemed acceptable according to the FDA guidance for industry (bioanalytical method validation). The FK-3000 concentration was measured in plasma samples up to 6 h following FK-3000 administration at an oral dose of 20 mg/kg. The findings indicate that the assay method is suitable for routine pharmacokinetic (PK) studies of FK-3000 in rats.

Simple RP-HPLC method for determination of triple drug combination of valsartan, amlodipine and hydrochlorothiazide in human plasma.

A simple RP-HPLC method for the quantification of valsartan (VAL), amlodipine (AML) and hydrochlorothiazide (HCT) in human plasma was developed and validated. VAL, AML and HCT were resolved using a Gemini C18 column and mobile phase gradient starting from 20 % acetonitrile and 80 % 10 mmol L(-1) ammonium formate (V/V, pH 3.5 ± 0.2, by formic acid) to 70 % acetonitrile and 30 % 10 mmol L(-1) ammonium formate, over 20 minutes, with a flow rate of 1 mL min(-1). The samples were purified by protein precipitation and extraction. Telmisartan was used as internal standard. The method was validated according to USFDA and EMEA guidelines with good reproducibility and linear responses R = 0.9985 (VAL), 0.9964 (AML), and 0.9971 (HCT). RSDs of intra- and inter-day precision ranged 2.2-8.1 and 4.6-11.7 %, respectively, for all three drugs. Mean extraction recoveries of three QCs for the triple drug combination were 76.5 (VAL), 72.0 (AML) and 73.0 (HCT) % for human plasma. Although the LC-MS/MS method is more sensitive than HPLC, HPLC is still suitable for preliminary pharmacokinetic study. The experiments performed demostrated that simultaneous determination of all components of the triple drug combination in human plasma can be done by this method. Proposed method can be also used for guidance to the LC-MS/MS method.