Journal of Chromatography B (v.869, #1-2)

This paper describes a new, fully automated on-line method combining restricted access material (RAM) extraction and ultra high-performance liquid chromatography (UHPLC) with mass spectrometric (MS) detection for determining congeners of bile acids (BAs) in human serum. In this method, low-pressure RAM and high-pressure UHPLC–MS are hyphenated by using a 2.5-mL loop-type interface. The compatibility problem between the large volume (1.2 mL) of strong solvent (methanol) used for RAM elution and the need for a weak solvent in UHPLC injection has been addressed by using an auxiliary pre-column cross-flow of 0.1% aqueous formic acid. In this way, the complete 2.5 mL loop volume can be injected into the UHPLC system, thereby maximizing sensitivity while maintaining good chromatographic performance. The optimised method allows the simultaneous analysis of 13 bile acids in a single run, including glycine- and taurine-conjugated bile acids, cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), ursodeoxycholic acid (UDCA), and litocholic acid. The complete analysis of a 100-μL single serum sample is performed in 30 min, providing detection limits in the pg range (corresponding with clinically relevant concentration levels) for all of the analytes except lithocholic acid, intra-day precision values (%R.S.D.) below 4% (except ursodeoxycholic acid) and inter-day precision lower than 15% (except ursodeoxycholic, glycoursodeoxycholic acid (GUDCA) and lithocholic acid).
Keywords: Bile acids; On-line method; Restricted access material; Ultra high-performance liquid chromatography; Loop-type interface; Auxiliary flow;

A rapid and accurate method for quantification of amobarbital and phenobarbital was developed using gas chromatography–mass spectrometry (GC–MS) without derivatization. Though the compounds measured without derivatization showed low sensitivity because of adsorption, addition of 3% formic acid to the solvent improved the sensitivity for the analytes. Taking account of matrix effect, solid-phase and liquid–liquid extraction from serum were examined. The correlation coefficients of the calibration curves were 0.9995 or better, and the accuracy and precision of intraday and interday assays were in line with Food and Drug Administration (FDA) criteria.
Keywords: Amobarbital; Phenobarbital; Formic acid; Gas chromatography–mass spectrometry;

A sensitive and specific liquid chromatography–tandem mass spectrometry method was developed and validated for the quantification of erucylphosphohomocholine (erufosine, ErPC3) in pharmacokinetic studies. Nine-fold deuterated ErPC3 was used as the internal standard. Following protein precipitation, reversed phase chromatography was performed. For analyte detection, electrospray ionization in the positive mode was applied. The mass transition m/z 504.4 > 139.1 was recorded for ErPC3, and the transition m/z 513.7 > 139.1 for the internal standard, respectively. Good linearity with a correlation coefficient >0.99 was found for the range of 0.48–15 mg/L ErPC3 in plasma (0.93–29.8 μM), the important range for clinical pharmacokinetic analysis. Interassay coefficients (n  = 10) of variation between 4.2% and 5.5% were found for ErPC3 pool samples with concentrations between 4.7 mg/L and 44.0 mg/L, respectively. The method has been used for analyses during a phase I clinical trial of ErPC3.
Keywords: Erufosine; Isotope dilution; Liquid chromatography–tandem mass spectrometry (LC–MS/MS);

Simultaneous glycosylation analysis of human serum glycoproteins by high-performance liquid chromatography/tandem mass spectrometry by Akira Harazono; Nana Kawasaki; Satsuki Itoh; Noritaka Hashii; Yukari Matsuishi-Nakajima; Toru Kawanishi; Teruhide Yamaguchi (20-30).
Changes in the glycosylation of some serum proteins are associated with certain diseases. In this study, we performed simultaneous site-specific glycosylation analysis of abundant serum glycoproteins by LC/Qq-TOF MS of human serum tryptic digest, the albumin of which was depleted. The glycopeptide peaks on the chromatogram were basically assigned by database searching with modified peak-list text files of MS/MS spectra and then based on mass differences of glycan units from characterized glycopeptides. Glycopeptide of IgG, haptoglobin and ceruloplasmin were confirmed by means of a comparison of their retention times and m/z values with those obtained by LC/MS of commercially available glycoproteins. Mass spectrometric carbohydrate heterogeneity in the assigned glycopeptides was analyzed by an additional LC/MS. We successfully demonstrated site-specific glycosylation of 23 sites in abundant serum glycoproteins.
Keywords: Glycosylation analysis; Human serum; Glycopeptide; LC/MS; LC/MS/MS; Immunoglobulin G; Haptoglobin; Transferrin; Ceruloplasmin;

A liquid chromatography–mass spectrometry (LC/MS) assay method was developed for the quantification of PSC 833 in rat plasma, using amiodarone as internal standard (IS). Separation was achieved using a C8 3.5 μm (2.1 mm × 50 mm) column heated to 60 °C with a mobile phase consisting of acetonitrile–ammonium hydroxide 0.2% (90:10 v/v) pumped at a rate of 0.2 mL/min. Detection was accomplished by mass spectrometer using selected ion monitoring (SIM) in positive mode. An excellent linear relationship was present between peak height ratios and rat plasma concentrations of PSC 833 ranging from 10 to 5000 ng/mL (R 2  > 0.99). Intra-day and inter-day coefficients of variation (CV%) were less than 15%, and mean error was less than 10% for the concentrations above the limit of quantification. The validated limit of quantification of the assay was 10 ng/mL based on 0.1 mL rat plasma. The method limit of detection, based on an average signal-to-noise (S/N) ratio of 3, was found to be 2.5 ng/mL. The assay was capable of measuring the plasma concentrations of PSC 833 in rats injected with a single dose of 5 mg/kg of the drug. PSC 833 and IS eluted within 4 min, free of interfering peaks. The method was found to be fast, sensitive, and specific for the quantification of PSC 833 in rat plasma.
Keywords: PSC 833; Valspodar; LC/MS; Pharmacokinetics;

A simple bioanalytical assay for determination of montelukast in human plasma: Application to a pharmacokinetic study by Pattana Sripalakit; Bungon Kongthong; Aurasorn Saraphanchotiwitthaya (38-44).
An analytical method based on high-performance liquid chromatographic (HPLC) was developed for the determination of montelukast in human plasma using mefenamic acid as an internal standard. After precipitation of plasma proteins with acetonitrile, chromatographic separation was carried out using a Zorbax Eclipse® XDB C8 (150 mm × 4.6 mm i.d., 5 μm) with mobile phase consisted of methanol–acetonitrile–0.04 M disodium hydrogen orthophosphate (22:22:56, v/v, pH 4.9). The wavelengths of fluorescence detection were set at 350 nm for excitation and 450 nm for emission. The linearity was confirmed in the concentration range of 5–1000 ng/ml in human plasma. Intra- and inter-day accuracy determined from quality control samples were 101.50 and 107.24%, and 97.15 and 100.37%, respectively. Intra- and inter-day precision measured as coefficient of variation were ≤4.72 and ≤9.00%, respectively. Extraction recoveries of drug from plasma were >48.14%. The protocol herein described was employed in a pharmacokinetic study of tablet formulation of montelukast in healthy Thai male volunteers.
Keywords: Montelukast; Assay; HPLC; Validation; Pharmacokinetics;

Isolation and characterization of degradation impurities in epirubicin hydrochloride injection by Dinesh Kumar; Rajesh Singh Tomar; Santosh Kumar Deolia; Rajesh Srivastava; Moloy Mitra; Surendera Tyagi (45-53).
The degradation of epirubicin hydrochloride aqueous formulation has been investigated during stability study. Some unknown degradation impurities were detected and out of these, three were characterized. These degradation impurities were isolated, enriched and were subjected to mass and NMR spectral studies. Based on the spectral data these were characterized as epirubicin dimer (impurity-1), 4-(4-amino-5-hydroxy-6-methyl-tetrahydro-pyran-2-yloxy)-2,5,12-trihydroxy-7-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydro-naphthacene-2-carboxylic acid hydroxymethyl ester (impurity-3) and 4-(4-amino-5-hydroxy-6-methyl-tetrahydro-pyran-2-yloxy)-2,5,12-trihydroxy-7-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydro-naphthacene-2-carboxylic acid (impurity-4). Structure elucidations of these degradation impurities are discussed in detail. Out of these degradation impurities, epirubicin dimer (impurity-1) has been previously identified while the other two impurity-3 and impurity-4 were previously unreported.
Keywords: Epirubicin; Degradation impurity; Identification; Chromatography; HPLC; Preparative LC; Lyophilization; Spectroscopy; Characterization;

Characterisation of protein composition and detection of IgA in cervicovaginal fluid by microchip technology by József Werling; Béla Kocsis; Diane Dean; Ildikó Kustos (54-58).
In this paper the application of microchip electrophoresis to examine the protein profile of cervicovaginal fluid and the detection of IgA heavy and light chains is presented. This method is a fast growing field of technology and ensures high-speed analysis requiring only microliters of sample. Proteins with wide range of molecular masses could be separated within 1 min. Cervicovaginal specimens of healthy women showed a complex protein pattern-containing several peaks in the 15–70 kDa region. sIgA is considered to be an important protein constituent of all mucosal surfaces. Detection of sIgA in cervicovaginal samples was achievable by microchip technology. Under reduced circumstances (induced by mercaptoethanol, a component of the denaturating solution) the disulfide bonds connecting IgA heavy and light chains are broken up and chains can be detected as separate peaks during electrophoresis. In 82.5% of the cases only the light chain of IgA could be detected in the clinical samples. The intact IgA heavy chain could be demonstrated in only 12.5% of the cases. Based on our data some conclusions were provided about the correlation of these patterns with the age of patients, pH of the cervicovaginal fluid, operations performed before sample collection and usage of oral contraceptives.
Keywords: Microchip electrophoresis; Cervicovaginal fluid; Protein; IgA;

A reversed-phase liquid chromatography method involving pre-column derivatisation with fluorescein isothiocyanate (FITC, isomer I) for determination of tobramycin in urine samples after inhalation has been developed. FITC reacts with the primary amino groups of tobramycin and other aminoglycosides under mild conditions to form a highly fluorescent and stable derivative. The chromatographic separation was carried out on a Phenomenex Luna C18 column at ambient temperature using a constant flow rate of 1 ml/min and mobile phase of acetonitrile–methanol–glacial acetic acid–water (420:60:5:515, v/v/v/v). The tobramycin–FITC derivative was monitored by fluorescent detection at an excitation wavelength 490 nm and emission wavelength 518 nm. The linearity of response for tobramycin was demonstrated at 11 different concentrations of tobramycin extracted from spiked urine, ranging from 0.25 to 20 μg/ml. Tobramycin and neomycin were extracted from spiked urine by a solid phase extraction clean-up procedure on a carboxypropyl-bonded phase (CBA) weak cation-exchange cartridge, and the relative recovery was >99% (n  = 5). The limit of detection (LOD) and limit of quantitation (LOQ) in urine were 70 and 250 ng/ml, respectively. The method had an accuracy of <0.2%, and intra-day and inter-day precision (in term of %coefficient of variation) were <4.89% and 8.25%, respectively. This assay was used for urinary pharmacokinetic studies to identify the relative lung deposition of tobramycin post-inhalation of tobramycin inhaled solution 300 mg/5 ml (TOBI®) by different nebuliser systems.
Keywords: Tobramycin; Fluorescein isothiocyanate (FITC); HPLC; Assay; Validation; Urinary pharmacokinetic;

The iodine-azide detection system to determine methimazole following its separation by RP-HPLC is described in this paper. The reaction between iodine and azide ions induced by methimazole was applied as a post-column reaction detection system. Neither extraction nor preconcentration of the sample was necessary. The methimazole standards added to normal urine show that the response of the detector, set at 350 nm (corresponding to unreacted iodine in the post-column iodine-azide reaction), was linear within the concentration range 2–10 nmol/mL of urine. The relative standard deviation values for precision and recovery within the calibration range were from 0.3 to 3.2% and from 97 to 102%, respectively. Limits of detection (LOD) and quantitation (LOQ) were 1 and 2 nmol/mL of urine, respectively. The method was applied to the separation and determination of patient urine samples and the analytical results were satisfactory.
Keywords: Iodine-azide reaction; Methimazole assay; Urine; HPLC;

Study of diosgenin-induced apoptosis kinetics in K562 cells by Sedimentation Field Flow Fractionation by J. Bertrand; B. Liagre; G. Bégaud-Grimaud; M.O. Jauberteau; P. Cardot; J.L. Beneytout; S. Battu (75-83).
Recently, the use of SdFFF in cancer research has been studied in order to better understand major phenomena implicated in cancer development and therapy: apoptosis and differentiation. In this report, we used SdFFF as a monitoring and cell separation tool to study the kinetics of apoptosis. Incubation of K562 cells with diosgenin, used as cellular model, led to a surprising apoptotic process occurring in two phases (after 24 and 48 h incubation), associated with specific p-ERK expression. Based on the capacity to sort apoptotic cells, results showed that SdFFF cell separation was an effective analytical tool to obtain different subpopulations regardless of the kinetics and extent of apoptosis. Results also showed that, after proper biological calibration of elution profiles, SdFFF can be used to monitor either the induction or the kinetics of a biological event.
Keywords: K562 cells; Diosgenin; Apoptosis; Sedimentation Field Flow Fractionation; Cell sorting;

A new and very sensitive analytical method has been developed and validated to jointly determine the anti-inflammatory drug ciclesonide (CIC), its active principle metabolite M1 (CIC-M1) and fluticasone propionate (FP) in human serum, in the low concentration range from 10 to 1000 pg/mL. This was accomplished by high-performance liquid chromatography and tandem mass spectrometry using atmospheric pressure photo ionisation (HPLC–MS/MS with APPI) using 0.5 mL of serum. Serum was mixed with the internal standards (IS) D11-CIC and D11-CIC-M1 and extracted with diisopropylether. A gradient with acetonitrile (containing 10 mM of acetic acid and 10% of acetone) was used. HPLC–MS/MS of the acetic acid adducts of the analytes was performed in negative mode. The novel aspect of this method is that instead of the dopant being introduced directly into the source by means of an external HPLC pump, it was added to the mobile phase. This provided significantly better sensitivity than the usual method of in-source addition of the dopant, and with no loss in HPLC performance. Sensitivity for the analytes was about four times greater than with either APCI or ESI. Validation was performed in three batches. The inter-batch precision (CV) of the quality control samples in human serum ranged from 4.08% to 6.78% for CIC, from 2.57% to 7.74% for CIC-M1, and from 2.38% to 9.61% for FP. The inter-batch accuracy (with reference to the mean value) of the quality control samples in human serum ranged from 99.3% to 110.0% for CIC, from 101.8% to 104.7% for CIC-M1, and from 100.4% to 101.8% for FP. Calibration data and LLOQ data are also presented in this paper. The analytes were stable in human serum over three freeze/thaw cycles, or for 4 h at room temperature, or for at least 18 months when stored at below −20 °C. This method was used for quantifying the analytes after inhalation of low-μg amounts of the drugs by patients.
Keywords: Ciclesonide; Fluticasone propionate; Human serum; Atmospheric pressure photo ionisation; Tandem mass spectrometry; Liquid chromatography;

Determination of biotin (vitamin H) by the high-performance affinity chromatography with a trypsin-treated avidin-bound column by Kou Hayakawa; Noriyuki Katsumata; Masahiko Hirano; Kazuyuki Yoshikawa; Tsutomu Ogata; Toshiaki Tanaka; Takeaki Nagamine (93-100).
A method for measuring biotin by affinity-chromatography was developed using a trypsin-treated avidin silica gel column. Elution was by a linear gradient of propan-2-ol in an acidic phosphate buffer system containing 0.7 M NaCl (pH 2.4). Biotin was derivatized with 9-anthryldiazomethane (ADAM) to the fluorescent biotin-ADAM ester and a linear calibration line was obtained from 0 to 1.39 pmol with a detection limit of 69.5 fmol. Biotin was measured after hydrolysis in 2.25 M sulphuric acid for 1 h at 120 °C and the recovery for biocytin was 65.7 ± 2.53%, and hence a correction factor of 1.52 was used for the total biotin analysis. The recovery of added biotin from the serum was more than 98% using this correction factor of 1.52. Biotin was also measured in nutritional supplemental foods and foodstuffs, and we found that chicken egg yolk, “natto”, rice bran, royal jelly, and dried yeast contained highest levels of biotin. Biotin was also found in ferments by Bacillus natto, yeast, and some acetic acid bacterium. Storage foods such as beans, nuts and eggs also contained abundant biotin. Biotin was also determined and replacement monitored in the serum of suspected biotinidase deficiency patients. This affinity-chromatographic method for biotin determination was shown to be a robust and reliable and is well suited for biochemical and nutritional research.
Keywords: Biotin; Vitamin H; High-performance affinity chromatograhy; Avidin; Trypsin-treated avidin; Total biotin; Free biotin; 9-anthryldiazomethane (ADAM); Fluorimetric method; Foodstuffs; Fermented foods; Body fluids; Recovery test;

A sensitive method for the simultaneous quantification of eight anticoagulant rodenticides (brodifacoum, bromadiolone, chlorophacinone, coumatetralyl, difenacoum, difethialone, flocoumafen and warfarin) in animal plasma and liver using liquid chromatography combined with heated electrospray ionization tandem mass spectrometry (LC-HESI-MS/MS) is described. The sample preparation includes a liquid–liquid extraction with acetone. The compound 7-acetoxy-6-(2,3-dibromopropyl)-4,8-dimethylcoumarin is used as an internal standard. Chromatographic separation was achieved using a Nucleodur C18 gravity column. Good linearity was observed up to 750 ng mL−1 for chlorophacinone and up to 500 ng mL−1 for the other compounds in plasma. In liver, good linearity was seen up to 500 ng g−1 for brodifacoum, chlorophacinone, difenacoum and difethialone and up to 750 ng g−1 for the other compounds. Depending on the compound, a level of 1 or 5 ng mL−1 could be quantified fulfilling the criteria for accuracy and precision and was therefore set as limit of quantification of the method in plasma. In liver, the limit of quantification was set at 250 ng g−1 for coumatetralyl and warfarin and at 100 ng g−1 for the other compounds. In plasma, the limit of detection varied from 0.07 ng mL−1 for flocoumafen to 3.21 ng mL−1 for brodifacoum. In liver, the limit of detection varied from 0.37 ng g−1 for warfarin to 4.64 ng g−1 for chlorophacinone. The method was shown to be of use in a pharmacokinetic study after single oral administration to mice and in the confirmation of suspected poisoning cases in domestic animals.
Keywords: Anticoagulant rodenticides; Multi-residue; Liquid chromatography heated electrospray ionization tandem mass spectrometry; Quantification; Plasma; Liver;

The purpose of this work was to study the stability of betahistine (BET) at different stress conditions and to develop a sensitive stability-indicating high-performance liquid chromatographic (HPLC) assay method. The stress conditions applied were including the effect of heat, moisture, acid–base, and ultra-violet (UV) light. Betahistine and its decomposition products were derivatized by reaction with dansyl chloride (Dan-Cl) and analyzed by HPLC equipped with fluorescence detector (FL) set at 336 and 531 nm as excitation and emission wavelengths, respectively. The drug was particularly labile at UV light and oxygen rich media. Two potential degradation products could be separated and identified by spectral methods. The chromatographic method involved Zorbax Eclipse XDB-C18 column kept at 30 ± 2 °C and a gradient elution with mobile phase composed of acetonitrile and 0.02 mol L−1 sodium acetate. The response factor of dansylated BET monitored by fluorescence detection was 32 times more than its UV response. The calibration curve of BET in bulk form was linear from 0.005 to 4.2 ng μL−1. Intraday and interday precision were less than 0.04% (CV), and accuracy was between 99.2% and 100.9% over 2.0 ng μL−1. The limit of detection was 0.002 ng μL−1. The method was also validated for sample stability during reaction, robustness and selectivity. The method was applied for purity testing of betahistine in tablet form.
Keywords: Betahistine; Betahistine N-oxide; Stability; Photodegradation; Dansyl chloride; TLC; HPLC;

A rapid and specific analytical method for simultaneous determination and quantification of seven major phospholipid classes in human blood was developed by normal-phase high-performance liquid chromatography tandem mass spectrometry. The optimal separation was achieved by using mobile phase hexane (A) and 2-propanol with water, formic acid and ammonia as modifiers (B) using an HPLC diol column. Isocratic elution method was used for better repeatability and no balance time. The seven major phospholipid classes in human blood that were detected including phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI) phosphatidylcholine (PC), lysophosphatidylcholine (Lyso-PC), and sphingomyelin (SM). That can be separated in this condition. Every phospholipid class contains many molecular species which have similar structure. The structure of phospholipids molecular species was identified by ion-trap MS n which produced ion fragments. And the qualification was completed by TOF-MS which shows good accuracy. Through the accurate quantification of one representative phospholipids molecule in each class, a method for simultaneous estimation hundreds of molecular species in seven major classes was established. The intra-day and inter-day precision and recovery had been investigated in detail. The RSD of precision for most compound is below 8% and RE is below 10%. Recovery is almost over 80%. This method was applied to phospholipids disorder related with diabetes nephropathy successfully. The concentrations of most phospholipids for normal people are higher than that for diabetic nephropathy (DN) patients in three phases. For most of phospholipids, with the development of DN the concentration was decreasing.
Keywords: Phospholipids; Quantification; Diabetes nephropathy; Normal-phase HPLC; Ion-trap MS n ; TOF-MS;

To evaluate the pharmacokinetics of digoxin in humans, a sensitive and specific LC/MS/MS method was developed and validated for the determination of digoxin concentrations in human plasma. The method was shown to be more sensitive, specific, accurate, and reproducible than common techniques such as RIA. For detection, a LC/MS/MS system with electro spray ionization tandem mass spectrometry in the positive ion-multiple reaction-monitoring (MRM) mode was used to monitor precursor to product ions of m/z 798.5–51.5 for digoxin and m/z 782.5–35.5 for the internal standard, digitoxin. The method was validated over a concentration range of 0.02–5 ng/mL and was found to have acceptable accuracy, precision, linearity, and selectivity. The mean extraction recovery from spiked plasma samples was above 80%. Imidafenacin, coadministered in a drug–drug interaction study, had no detectable influence on the determination of digoxin in human plasma. The novel method was applied to a drug–drug interaction study of digoxin and imidafenacin and the characterization of steady-state pharmacokinetics of digoxin in humans after oral administration at a dose of 0.25 mg on days 1 and 2 followed by 0.125 mg daily doses on days 3 through 8.
Keywords: Digoxin; LC/MS/MS; 96-Well SPE; Human plasma; Imidafenacin;

Iodixanol is a widely used iso-osmolar contrast medium agent. Similar to iohexol, it can also be a good exogenous marker for the measurement of glomerular filtration rate (GFR). This article describes the development and validation of an HPLC-UV method for quantification of iodixanol in human plasma. Internal standard, iohexol (20 μl, 1 mg/ml), and perchloric acid (30 μl, 20%, v/v) were added to plasma samples (300 μl), followed by neutralization with 10 μl potassium carbonate (5 M). Samples were centrifuged and 10 μl of the supernatant was injected onto a C18 EPS analytical column (3 μm particle size, 150 mm × 4.6 mm). The extraction method yielded >95% recovery for both iodixanol and iohexol. The mobile phase consisted of 0.1% (w/v) sodium formate buffer and acetonitrile. Iohexol and iodixanol peaks were eluted at ∼5 and 9 min, respectively using a fast gradient method. The assay lower limit of detection was 2.0 μg/ml and lower limit of quantification was 10 μg/ml. The calibration curves, assessed in six replicates, were linear over an iodixanol concentration range of 10–750 μg/ml. Intra- and inter-day accuracy was >95% and precision expressed as % coefficient of variation was <10%. This method is simple, accurate, precise and robust and can potentially be used for iodixanol quantification in large-scale clinical studies.
Keywords: Assay; Concentration; Contrast media agent; GFR; HPLC-UV; Iodixanol; Iohexol; Kidney function;

A simple and sensitive HPLC method has been developed and validated for the determination of oridonin (ORI) in rabbit plasma. A simple liquid–liquid extraction (LLE) method was applied to extract ORI and the internal standard (IS), isopsoralen, from rabbit plasma. Chromatographic separation of ORI and the IS was achieved with a Kromasil C18 5-μm column (250 mm × 4.6 mm) using methanol–water (50:50, v/v) as mobile phase at a flow rate of 1 mL/min. The ultraviolet (UV) detection wavelength was set at 241 nm. The lower limit of quantification (LLOQ) was 0.02 μg/mL. The calibration curves were linear over a concentration range of 0.02–10 μg/mL. The assay accuracy and precision were within the range of 95.1–113.5% and 5.4–8.6%, respectively. This HPLC method was applied successfully to the pharmacokinetic study of ORI-loaded poly(caprolactone)-poly(ethylene oxide)-poly(caprolactone) copolymer nanoparticles (ORI-PCL-PEO-PCL-NP) in rabbits, given as a single intravenous injection at the dose equivalent to 2 mg of ORI/kg, and the pharmacokinetic parameters for ORI were compared with a single intravenous injection of a ORI solution at the same dose.
Keywords: Oridonin; HPLC; Pharmacokinetics; Nanoparticles;

Determination of salirasib (S-trans,trans-farnesylthiosalicylic acid) in human plasma using liquid chromatography–tandem mass spectrometry by Ming Zhao; Ping He; Linping Xu; Manuel Hidalgo; Dan Laheru; Michelle A. Rudek (142-145).
A liquid chromatography/tandem mass spectrometric (LC/MS/MS) assay was developed for the quantitative determination of salirasib (S-trans,trans-farnesylthiosalicylic acid, FTS) in human plasma. Sample pretreatment involved liquid–liquid extraction with methyl t-butyl ether of 0.5-mL aliquots of lithium heparin plasma spiked with the internal standard, S-trans,trans-5-fluoro-farnesylthiosalicylic acid (5-F-FTS). Separation was achieved on Waters X-Terra™ C18 (50 mm × 2.1 mm i.d., 3.5 μm) at room temperature using isocratic elution with acetonitrile/10 mM ammonium acetate buffer mobile phase (80:20, v/v) containing 0.1% formic acid at a flow rate of 0.20 mL/min. Detection was performed using electrospray MS/MS by monitoring the ion transitions from m/z 357.2 → 153.0 (salirasib) and m/z 375.1 → 138.8 (5-F-FTS). Calibration curves were linear in the concentration range of 1–1000 ng/mL. A 5000 ng/mL sample that was diluted 1:10 (v/v) with plasma was accurately quantitated. The values for both within day and between day precision and accuracy were well within the generally accepted criteria for analytical method (<8.0%). This assay was subsequently used for the determination of salirasib concentrations in plasma of cancer patients after oral administration of salirasib at a dose of 400 mg.
Keywords: Salirasib; LC/MS/MS; Pharmacokinetics;