Journal of Chromatography B (v.827, #2)

Verapamil quantification in human plasma by liquid chromatography coupled to tandem mass spectrometry by Ney Carter do C. Borges; Gustavo D. Mendes; Rafael E. Barrientos-Astigarraga; Paulo Galvinas; Celso H. Oliveira; Gilberto De Nucci (165-172).
An analytical method based on liquid chromatography with positive ion electrospray ionization (ESI) coupled to tandem mass spectrometry detection (LC–MS/MS) was developed for the determination of Verapamil in human plasma using Metoprolol as the internal standard. The analyte and internal standard were extracted from the plasma samples by liquid–liquid extraction and chromatographed on a C8 analytical column. The mobile phase consisted of methanol–water (70:30; v/v) + 12 mM formic acid. The method had a chromatographic total run time of 3.5 min and was linear within the range 1.00–500 ng/mL. Detection was carried out on a Micromass Quattro Ultima tandem mass spectrometer by multiple reaction monitoring (MRM). The intra-run imprecision was less than 5.1% calculated from the quality control (QC) samples, and 16.3% from the limit of quantification (LOQ). The accuracy determined from QC samples were between 92.9 and 103.1%, and 95.2 and 115.3% from LOQ. Concerning the inter-batch analysis, the imprecision was less than 5.8% and 17.3% from QC samples and LOQ, respectively. The accuracy varied between 98.2 and 100.8% from QC and it was 103.1% from LOQ. The protocol herein described was employed in a bioequivalence study of two tablet formulations of Verapamil.
Keywords: Verapamil; Bioequivalence; LC–MS/MS; Metoprolol;

Magnesia–zirconia based mimetic biomembrane chromatography for predicting human drug absorption by Zhi-Xiong Hu; Wei-Nong Zhang; Hai-Bo He; Yu-Qi Feng; Shi-Lu Da (173-181).
In this paper, a novel mimetic biomembrane chromatography stationary phase of magnesia–zirconia composite matrix were prepared with the Lewis acid–base interaction between phosphatidylcholine's residue phosphonate group and Lewis acid sites of magnesia–zirconia composite; the retention factors of a chemically diverse set of drugs on the new stationary phase were determined; the drugs log  K mbm values were correlationed with the absorbed fraction of drugs orally administered in humans (%F a) and a hyperbolic relationship was obtained. Meanwhile, the relationship between the log  K mbm values and hydrophobic parameters (log  P oct and log  D oct) were discussed. The usefulness of the new column for predicting oral drug absorption in humans is demonstrated by comparing this model with IAM, ILC and BMC models. Results show that the log  K mbm values have good relationship with log K W IAM , log  K BMC and have moderate to fair relationship with log  K s determined on four different ILC column (EPL, PC, PC-PE, PC-PS). Therefore, the log  K mbm values can provide key information about the transport properties of drugs and this chromatographic model may be applicable for prediction of drug uptake through epithelial cell membranes during the drug discovery process.
Keywords: Magnesia–zirconia composite; Oral drug absorption; Mimetic biomembrane stationary phase;

A generic affinity chromatography purification protocol for the isolation of preparative quantities of pure and stable polyclonal antibodies to hydrophobic haptenic analytes is described together with a panel of tests to monitor the purification process and assess the functional and structural purity of isolated antibodies. The purification method is based on the use of a mixture of acetonitrile and propionic acid to elute bound antibodies from Sepharose 4B-based immunoabsorbent gels. Highly specific and pure antibodies to steroid estrogens, pentachlorophenol and Irgarol 1051 were isolated in 50–150 mg quantities per preparation in a batch-wise method using appropriate ligands linked to the solid phase via a hydrophilic chemical arm, tetraethylene pentamine. The panel of ELISA tests together with SDS–PAGE enabled the monitoring of the absorption and elution steps and provided data relevant to the assessment of the degree of structural and functional purity of the isolated antibody preparations. The study demonstrates that the affinity purification procedure is practical, simple, generic for antibodies to hydrophobic haptens and suitable for scaling up. In addition, the study showed that the functional properties of the affinity-purified antibodies indicated improvements on the operational properties (specificity and assay detection limits) of the source antisera. The isolated IgG antibodies showed near 100% functional and structural purity and no deterioration of activity on storage for long periods. The method provides critical reagents for labelled-antibody immunoassays and immunosensors and antibody-dependent sample purification techniques.
Keywords: Immunoaffinity chromatography; Hydrophobic haptens; Generic elution methods;

We have compared two sample preparation methods for the analysis of plasma acylcarnitines by tandem mass spectrometry. Extraction from liquid plasma using acetonitrile was compared with the widely used methanol extraction from plasma spotted on filter paper. The recovery and reproducibility of the acetonitrile extraction were improved by acidification with 0.3% formic acid. The acidified acetonitrile and methanol extractions have the same limit of detection and upper linearity limit for all acylcarnitine species studied. The correlation coefficients between the two methods were greater than 0.988 and the slopes of the linear regressions ranged from 0.901 to 1.070. The extraction of acylcarnitines by acidified acetonitrile from liquid plasma yielded results comparable to those obtained by methanol extraction from plasma spotted on filter paper.
Keywords: Acylcarnitine analysis; Organic acidemia; Defects of fatty acid oxidation; Inherited disorders; Tandem mass spectrometry;

Metabolism of isometheptene in human urine and analysis by gas chromatography–mass spectrometry in doping control by Emmanouil Lyris; George Tsiakatouras; Yiannis Angelis; Michael Koupparis; Maria-Helen Spyridaki; Costas Georgakopoulos (199-204).
A study of the metabolism of isometheptene, an antispasmodic drug, in man and comparison with heptaminol metabolism, is presented in this paper. Isometheptene and two metabolites were detected in human urine after oral administration of a tablet containing isometheptene mucate. The urine level of the parent drug, which is excreted during the first 24 h, was determined using gas chromatography–mass spectrometry, after alkaline extraction with organic solvent. A minor metabolite of isometheptene was converted to heptaminol in vitro under the acidic hydrolysis conditions used for the screening procedure of stimulants and narcotics in doping control analysis.
Keywords: Doping control; Isometheptene; Gas chromatography–mass spectrometry; Heptaminol;

A sensitive and selective LC–MS–MS method has been developed and validated for the determination of cryptotanshinone (CTS) and its active metabolite tanshinone II A (TS II A) in rat plasma using fenofibrate (FOFB) as internal standard. Liquid–liquid extraction was used for sample preparation. Chromatographic separation was achieved on a Waters symmetry ODS column using methanol and water (85:15) as mobile phase delivered at 1.0 mL/min. LC–MS–MS analysis was carried out on a Finnigan LC-TSQ Quantum mass spectrometer using atmospheric pressure chemical ionization (APCI) and positive multiple reaction monitoring. Ions monitored were m/z 297.0 → 251.0 for CTS, m/z 295.0 → 249.0 for TS II A, and m/z 361.1 → 233.0 for FOFB with argon at a pressure of 0.2 Pa and collision energy of 25 eV for collision-induced dissociation (CID). The assay was linear over the range 0.1–20 ng/mL for CTS and 0.2–15 ng/mL for TS II A. The average recoveries of CTS and TS II A from rat plasma were 93.7 and 94.7%, respectively. The established method has been applied in a pharmacokinetic study of CTS in rats.
Keywords: Cryptotanshinone; Tanshinone II A; Metabolism; Pharmacokinetic; LC–MS–MS;

Analysis of creatine, creatinine, creatine-d3 and creatinine-d3 in urine, plasma, and red blood cells by HPLC and GC–MS to follow the fate of ingested creatine-d3 by Lauren MacNeil; Lisa Hill; Daniel MacDonald; Lori Keefe; James F. Cormier; Darren G. Burke; Truis Smith-Palmer (210-215).
Creatine, which is increasingly being used as an oral supplement, is naturally present in the body. Studies on the fate of a particular dose of creatine require that the creatine be labeled, and for studies in humans the use of a stable isotopic label is desirable.The concentrations of total creatine and total creatinine were determined using HPLC. Creatine and creatinine were then separated using cation exchange chromatography and each fraction was derivatized with trifluoroacetic anhydride and the ratio of the deuterated:undeuterated species determined using GC–MS. Ratios of creatine:creatine-d3, and creatinine:creatinine-d3, and the concentrations of each of these species, were able to be determined in urine, plasma and red blood cells. Thus, the uptake of labeled creatine into plasma and red blood cells and its excretion in urine could be followed for a subject who ingested creatine-d3. Creatine-d3 was found in the plasma and red blood cells 10 min after ingestion, while creatine-d3 and creatinine-d3 were found in the urine collected after the first hour.
Keywords: Creatine; GC–MS; Deuterated creatine; Deuterated creatinine; Urine; Plasma; Red blood cells;

A simple, rapid, sensitive and reproducible method based on solid-phase extraction (SPE) and acidified silica clean-up was developed for the measurement of 12 polybrominated diphenyl ethers (PBDEs), including BDE 209, and 2,2′,4,4′,5,5′-hexabromobiphenyl (BB 153) in human serum. Several solid-phase sorbents (Empore™ C18, Isolute Phenyl, Isolute ENV+ and OASIS™ HLB) were tested and it was found that OASIS™ HLB (500 mg) gives the highest absolute recoveries (between 64% and 95%, R.S.D. < 17%, n  = 3) for all tested analytes and internal standards. Removal of co-extracted biogenic materials was performed using a 6 ml disposable cartridge containing (from bottom to top) silica impregnated with sulphuric acid, activated silica and anhydrous sodium sulphate. PBDEs and BB 153 were quantified using a gas chromatograph coupled with a mass spectrometer (MS) operated in electron-capture negative ionization mode. The method limits of quantification (LOQ) ranged between 0.2 and 25 pg/ml serum (0.1 and 4 ng/g lipid weight). LOQs were dependent on the analyte levels in procedural blanks which resulted in the highest LOQs for PBDE congeners found in higher concentrations in blanks (e.g. BDE 47, 99 and 209). The use of OASIS™ HLB SPE cartridge allowed a good method repeatability (within- and between-day precision < 12% for all congeners, except for BDE 209 < 17%, n  = 3). The method was applied to serum samples from a random Belgian population. The obtained results were within the range of PBDE levels in other non-exposed population from Europe.
Keywords: Polybrominated diphenyl ethers; Human serum; Solid-phase extraction;

The electrochemical (EC) detection of iodide at gold, silver and platinum electrodes under similar experimental conditions was evaluated. To achieve optimal amperometric detection, the electrode sensitivity, selectivity, and stability was compared. Isocratic separation of iodide was attained by ion chromatography (IC) using an anion-exchange column with nitrate as an eluent ion (25 mM HNO3  + 50 mM NaNO3). Although the Ag electrode showed the highest selectivity due to the relatively low applied potential (+0.10 V versus Ag|AgCl), it requires continuous surface polishing upon injection of standard solutions or real samples; in addition, the chromatographic peak of iodide exhibited a pronounced dip-tailing. The limit of detection (LoD) of iodide was estimated to be 3.5 μg/L (S/N = 3) with an injection volume of 50 μL. Likewise, pulsed electrochemical detection at the silver electrode did not demonstrate the expected results in terms of peak shape and low detection limit. Using the same chromatographic conditions, iodide detection at the Au electrode (E app  = +0.80 V versus Ag|AgCl) exhibited a regular peak shape accompanied by a sensitivity comparable to the silver one. Yet, upon continuous injections the signal intensity displayed a progressive lowering up to ca. 40% in 6 h. Best results in terms of signal stability, peak shape and analytical response were obtained with a modified platinum electrode which allowed to achieve a LoD of 0.5 μg/L (S/N = 3). The present IC–EC detection method using a modified Pt electrode (E app  = +0.85 V versus Ag|AgCl) was successfully applied to determine low contents of iodide in human urine with solid phase extraction as pretreatment. Such a developed method correlated very well with the reference colorimetric method in urine (r  = 0.95273), and it is specifically suggested when the iodide content is relatively low, i.e., <20 μg/L.
Keywords: Iodine; Ion chromatography; Electrochemical detection; Human urine;

Author Index (232-233).

Keyword Index (234-239).