Journal of Chromatography B (v.821, #1)

Determination of midazolam and its major metabolite 1′-hydroxymidazolam by high-performance liquid chromatography-electrospray mass spectrometry in plasma from children by Simon N. Muchohi; Steve A. Ward; Louise Preston; Charles R.J.C. Newton; Geoffrey Edwards; Gilbert O. Kokwaro (1-7).
We have developed a sensitive, selective and reproducible reversed-phase high-performance liquid chromatography method coupled with electrospray ionization mass spectrometry (HPLC–ESI-MS) for the simultaneous quantification of midazolam (MDZ) and its major metabolite, 1′-hydroxymidazolam (1′-OHM) in a small volume (200 μl) of human plasma. Midazolam, 1′-OHM and 1′-chlordiazepoxide (internal standard) were extracted from alkalinised (pH 9.5) spiked and clinical plasma samples using a single step liquid–liquid extraction with 1-chlorobutane. The chromatographic separation was performed on a reversed-phase HyPURITY™ Elite C18 (5 μm particle size; 100 mm × 2.1 mm i.d.) analytical column using an acidic (pH 2.8) mobile phase (water–acetonitrile; 75:25% (v/v) containing formic acid (0.1%, v/v)) delivered at a flow-rate of 200 μl/min. The mass spectrometer was operated in the positive ion mode at the protonated-molecular ions [M + l]+ of parent drug and metabolite. Calibration curves in spiked plasma were linear (r 2  ≥ 0.99) from 15 to 600 ng/ml (MDZ) and 5–200 ng/ml (1′-OHM). The limits of detection and quantification were 2 and 5 ng/ml, respectively, for both MDZ and 1′-OHM. The mean relative recoveries at 40 and 600 ng/ml (MDZ) were 79.4 ± 3.1% (n  = 6) and 84.2 ± 4.7% (n  = 8), respectively; for 1′-OHM at 30 and 200 ng/ml the values were 89.9 ± 7.2% (n  = 6) and 86.9 ± 5.6% (n  = 8), respectively. The intra-assay and inter-assay coefficients of variation (CVs) for MDZ were less than 8%, and for 1′-OHM were less than 13%. There was no interference from other commonly used antimalarials, antipyretic drugs and antibiotics. The method was successfully applied to a pharmacokinetic study of MDZ and 1′-OHM in children with severe malaria and convulsions following administration of MDZ either intravenously (i.v.) or intramuscularly (i.m.).
Keywords: Midazolam; 1′-Hydroxymidazolam; Pharmacokinetics in children;

High performance liquid chromatographic (HPLC) methods were validated for the determination of aripiprazole (OPC-14597, Abilify™) in rat plasma and brain. Separation was by Nova-pak phenyl column; flow rate, 1.0 ml/min; mobile phase, acetonitrile–methanol–20 mM sodium sulfate–acetic acid (27:25:48:1, v/v/v/v); UV detection at 254 nm. Reproducibility in plasma and brain showed excellent precision (within 7.8 and 10.6%) and accuracy (96.0–102.4% and 99.0–108.7%) with calibration curve ranges 10.0–2000 ng/ml and 30.0–6000 ng/g, respectively. Validated HPLC methods were successfully applied to pharmacokinetic study of aripiprazole in rats, demonstrating brain concentrations after oral administration five times higher than plasma concentrations.
Keywords: Aripiprazole; OPC-14597; Abilify™;

SPE-HPLC determination of new tetrahydroisoquinoline derivatives in rat plasma by M. Rizzo; D. Ventrice; G. De Sarro; R. Gitto; R. Caruso; A. Chimirri (15-21).
Recently a novel class of non-competitive AMPA receptor (AMPAR) antagonists, such as, N-acetyl-1-(p-chlorophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (PS3Ac) have been developed using molecular modeling studies. In this study we present a validated method for detecting PS3Ac in biological matrices by high performance liquid chromatography with ultraviolet detection. In this study PS3Ac was administered to Wistar rats. After intraperitoneal administration, the plasma concentrations of PS3Ac and its potential metabolic products, i.e., PS3OH, PS3 and PS3OHAc were determined. Serum samples (0.5 ml) were purified by solid-phase extraction of analytes using Oasis cartridges. The chromatographic separation was performed on a LiChrosorb RP-1 at 30 °C. The eluent was made of potassium dihydrogen phosphate/acetonitrile in ratio of 50:50 (v/v); the flow rate was 1 ml/min. The detection was performed at 220 nm. The method exhibited a large linear range from 0.05 to 5 μg/ml for all studied compounds. The intra-assay accuracy ranged from 92% determined at 0.1 μg/ml of PS3OH, to 108% determined at 0.05 μg/ml of PS3OHAc. The average coefficient of variation of inter-assay was 6.27%. The average recovery from plasma was 78.5%. The limits of quantification for all the tetrahydroisoquinoline derivatives was 20 ng. The method proved to be highly sensitive and specific for the determination of the studied compounds in rat plasma and has been successfully applied to the evaluation of the pharmacokinetic profile of the inoculated compound.
Keywords: AMPA receptor antagonist; SPE-HPLC; Pharmacokinetic;

LC/MS/MS measurement of gentamicin in bovine plasma, urine, milk, and biopsy samples taken from kidneys of standing animals by David N. Heller; James O. Peggins; Cristina B. Nochetto; Michelle L. Smith; O. Alberto Chiesa; Keesla Moulton (22-30).
Methods for the measurement of gentamicin concentration in several bovine tissues were developed and validated. A novel liquid chromatographic (LC) technique employed trifluoroacetic acid in the mobile phase so that all gentamicin components co-eluted. Analytes were ionized by positive-ion pneumatically assisted electrospray and detected by selected reaction monitoring (SRM) with an LC-tandem mass spectrometer (LC/MS/MS). Calibration of plasma and urine samples was based on tobramycin internal standard. Calibration of milk and kidney samples was based on external standard, due to variability of tobramycin response in these matrices. The extraction technique employed treatment with aqueous trichloroacetic acid to both precipitate protein and liberate gentamicin from the matrix. Milk samples had to be defatted by centrifugation prior to extraction. Urine samples were further cleaned up with C-18 solid phase extraction (SPE). These methods were validated for use in several residue depletion studies (reported elsewhere) to monitor the depletion of gentamicin in tissues under various dosing conditions. The plasma method was calibrated from 1 to 5000 ng/mL in two ranges, with a limit of quantitation (LOQ) in the low range calculated at 3.3 ng/mL. The milk method was calibrated from 2.5 to 2500 ng/mL with an LOQ calculated at 4.5 ng/mL. The urine method was designed for use at low levels, and was calibrated from 1 to 100 ng/mL with an LOQ of 3.8 ng/mL. The kidney method was primarily designed for analysis of small samples (approximately 100 mg). This method was calibrated from 10 to 50,000 ng/g with an LOQ of 26 ng/g.
Keywords: Gentamicin; Liquid chromatography-tandem mass spectrometry; Residue analysis; Quantification; Plasma; Urine; Kidney; Milk;

A simple, sensitive and specific liquid chromatography coupled electrospray ionization mass spectrometric (LC/ESI/MS) method for the determination of 13-O-demethylated metabolite (MI), one of the major metabolites of tacrolimus has been developed. The assay uses 32-demethoxyrapamycin (IS) as the internal standard; ethyl acetate as extraction solvent; a Hypersil-Keystone Beta Basic-18 reversed-phase column; and a gradient mobile phase of consisting 0.1% formic acid in water and methanol–acetonitrile (3:49, v/v). Mass detection is performed on a single quadrupole mass spectrometer equipped with an electrospray ionization (ESI) interface and operated in a positive ionization mode. MI in the microsomal incubates was quantitated by computing the peak area ratio (MI/IS) analyzed in single ion monitoring (SIM) mode (m/z: 804 and m/z: 901 for MI and IS, respectively). Precision of the assay was determined by calculating the intra-run and inter-run variation at three concentrations (15, 25, 80 ng/ml); the intra run relative standard deviation (R.S.D.) was less than 10% and ranged from 5.0 to 8.3%; and the inter-run R.S.D. was less than 10% and ranged from 4.6 to 9.6%. The limits of detection was 2 ng/ml. This assay has been used to evaluate the effect of three human immunodeficiency virus (HIV) protease inhibitors on the metabolism of tacrolimus in human liver microsomes.
Keywords: Tacrolimus; 13-O-demethyl tacrolimus; LC/ESI/MS; Human liver microsome; Protease inhibitors;

Separation and detection of vitellogenin in fish plasma by capillary zone electrophoresis by Maoyong Song; Jiangning Wang; Jing Shao; Bin He; Guibin Jiang; Guoqing Shi (38-44).
A method for coupling capillary zone electrophoresis (CZE) with rapid membrane chromatography purification (RMCP) was established for the analysis of vitellogenin (VTG) in male fish plasma induced with 17ß-estrodiol. CZE analyses of purified VTG were performed in a buffer containing 25 mM sodium borate (pH 8.4). A 50 μm i.d. fused-silica capillary was used for separation and the detection was carried out by UV-diode array at 214 nm. Inter- and intra-assay variabilities of the proposed method were less than 10.06 and 1.95%, respectively. The method has good linear relationship over the scope of 15–2250 μg/ml with a correlation coefficient of R 2  = 0.9965 and a detection limit of 7.0 μg/ml. The established CZE method was also applied to directly separate and identify VTG from fish plasma. The results indicated this method could minimize interferences from plasma proteins, allowing the detection of at least 62.5 μg/ml of VTG proteins in total proteins. This is a rapid and easy method to determine the quantity and purity of VTG compared to Bradford method and SDS-PAGE.
Keywords: Capillary zone electrophoresis; Vitellogenin; Fish plasma;

HPLC and tandem detection to monitor conformational properties of biopharmaceuticals by Dion M.A.M. Luykx; S.S. Goerdayal; P.J. Dingemanse; W. Jiskoot; Peter M.J.M. Jongen (45-52).
High-performance liquid chromatography (HPLC) with UV, circular dichroism (CD) and intrinsic fluorescence detection was applied to monitor conformational properties of recombinant human interferon α2b when performing size exclusion chromatography (SEC) and reversed-phase HPLC (RP-HPLC). In this way native conditions during SEC and structural changes of the protein during RP-HPLC were demonstrated. These results were confirmed by stand-alone fluorescence and CD measurements. With respect to HPLC tandem detection, the fluorescence detector compared favourably to the UV and CD detector regarding linearity, sensitivity and selectivity. SEC combined with intrinsic fluorescence scanning detection permits conformational analysis of small amounts of aggregates in the presence of excess native monomeric protein. In conclusion, HPLC with on-line UV and intrinsic fluorescence detection provides a promising concept for analysing the amount and conformational properties of a biopharmaceutical and its impurities.
Keywords: (Reversed-phase) HPLC; Biopharmaceuticals; Protein conformation; Intrinsic fluorescence; Circular dichroism; Tandem-detection; Interferon-alfa; Human growth hormone; Anti tetanus immunoglobulin; Human serum albumin;

Capillary electrophoretic (CE) method was developed for the determination of urinary 3-methylhistidine (3MH) and 1-methylhistidine (1MH) indicating the extent of degradation of skeletal muscle proteins and thereby the state of human health. 3MH, 1MH and histidine can be separated in both acidic and alkaline media, where these amino acids form cation and anion, respectively. The effective mobility of all ionic forms was measured over a broad range of pH (1.67–11.80), which made it possible to evaluate the corresponding dissociation constants. 3MH and 1MH were determined together with creatinine in untreated urine samples with the limit of detection of 2.4 μM (0.4 mg L−1) and 3.0 μM (0.5 mg L−1), respectively. Determination was fast and took ca. 12 min including the column washing. Method was employed for an analysis of urine collected from healthy individuals, and from the patients hospitalized with obesity and diabetes mellitus II. This analysis has revealed differences between the healthy individuals and the patients pointing to a more extensive degradation of muscle proteins in the latter group.
Keywords: 3-Methylhistidine; Capillary electrophoresis; Mobility; Untreated urine;

The partitioning of chymosin (from Aspergilus niger) and pepsin (from bovine stomach) was carried out in aqueous-two phase systems formed by polyethyleneglycol-potassium phosphate. The effects of polymer concentration, molecular mass and temperature were analysed. The partition was assayed at pH 7.0 in systems of polyethyleneglycol of molecular mass: 1450, 3350, 6000 and 8000. Both proteins showed high affinity for the polyethyleneglycol rich phase. The increase of polyethyleneglycol concentration favoured the protein transfer to the top phase, suggesting an important protein–polymer interaction. Polyethyleneglycol proved to have a stabilizing effect on the chymosin and pepsin, increasing its protein secondary structure. This finding agreed with the enhancement of the milk clotting activity by the polyethyleneglycol. The method appears to be suitable as a first step for the purification of these proteins from their natural sources.
Keywords: Pepsin; Chymosin; Partition; Acid protease;

Fingerprint analysis of Psoralea corylifolia L. by HPLC and LC–MS by Luhua Zhao; Chaoyu Huang; Zhen Shan; Bingren Xiang; Linghua Mei (67-74).
High-performance liquid chromatography (HPLC) was developed for fingerprint analysis of Psoralea corylifolia. Liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS n ) technique was first employed to identify the components of the fingerprint. The samples were separated with an Alltima C18 column (250 mm × 4.6 mm, 5 μm) by linear gradient elution using water–acetic acid (A; 100:0.1, v/v) and acetonitrile (B; 0 min, 40%; 15 min, 50%; 35 min, 60%; 45 min, 70%; 55 min, 80%; and maintained for 5 min) as mobile phase at a flow rate of 1.0 ml/min and detector wavelength at 245 nm. A standard procedure was developed for HPLC fingerprint analysis. Average chromatogram of 10 batches of P. corylifolia L. from Sichuan and Henan Provinces, PR China, which has been considered as the original and genuine herbal medicine for a long time, was first established as the characteristic fingerprint. There are 12 common peaks in this fingerprint. Ten of these common peaks were identified by MS data. This profile was then used to identify and assess the differences among the herb grown in various areas of China. The HPLC fingerprint analysis is specific and may serve for quality identification and comprehensive evaluation of P. corylifolia.
Keywords: Fingerprint; Psoralea corylifolia; HPLC; LC–MS; MS data; Identification;

Stability, pKa and plasma protein binding of roscovitine by Marina Vita; Mohamed Abdel-Rehim; Christina Nilsson; Zuzana Hassan; Patrik Skansen; Hong Wan; Lennart Meurling; Moustapha Hassan (75-80).
In the present investigation, the binding of roscovitine (100, 500 and 1500 ng/mL) to plasma proteins was studied at 25 and 37 °C by ultrafiltration and equilibrium dialysis methods. Drug stability in plasma was assessed during a 48 h at 4, 25 and 37 °C. The effect of thawing and freezing on drug stability was studied. The pKa of roscovitine was measured using capillary electrophoresis coupled with mass spectrometry. Roscovitine was quantified utilizing liquid chromatography and tandem mass spectrometry. Roscovitine is highly bound to plasma proteins (90%). Binding of roscovitine to human serum albumin was constant (about 90%) within concentration range studied while the binding to α1-acid glycoprotein decreased with increasing drug concentration indicating that albumin is more important in clinical settings. However, α1-acid glycoprotein might be important when plasma proteins change with disease. Protein binding was higher at 25 °C compared to 37 °C. The results obtained by equilibrium dialysis were in good agreement with those obtained by ultrafiltration. Roscovitine was stable at all temperatures studied during 48 h. Roscovitine has a pKa of 4.4 showing that the drug mainly acts like a weak mono-base. The results obtained in our studies are important prior to clinical trials and to perform pharmacokinetic studies.
Keywords: LC–MS–MS; Equilibrium dialysis; Ultrafiltration; pKa; Olomoucine; α1-Acid glycoprotein;

A matrix developed from N,N,N′,N′-ethylenediaminetetramethylenephosphonic acid-modified zirconia beads (further referred to as r_PEZ); 25–38 μm in diameter and with a pore size of 22 ± 3 nm, was utilized for the separation of immunoglobulins (Igs). r_PEZ has been shown to bind to various Igs originating from a wide variety of species. To understand the mechanisms controlling the uptake of Igs by r_PEZ, static protein uptake experiments were carried out. The protein uptake profiles were further modeled with a kinetic rate constant model. Individual studies were undertaken for human immunoglobulin A, G and M (HIgA, HIgG and HIgM). The kinetic rate constant model indicated that HIgG binding to r_PEZ was more favorable than its disassociation. The equilibrium rate constants were found to decrease with increasing concentration. The effect of continuous loading in a packed bed system utilizing r_PEZ matrix was evaluated by carrying out frontal studies, using different feed concentrations and linear velocities. The breakthrough profiles obtained for the uptake of HIgG were modeled with the pore diffusion model. The model was found to best describe the breakthrough profiles obtained at a feed concentration of 2.0 mg of HIgG per milliliter. The NTU for the packed bed was found to be equal to 2.
Keywords: Zirconia; Immunoglobulins; Pseudo-affinity separations; Modeling;

A simple liquid chromatographic method based on intramolecular excimer-forming derivatization and fluorescence detection for the determination of tyrosine and tyramine in urine by Hideyuki Yoshida; Hitoshi Nohta; Yumiko Harada; Makoto Yoshitake; Kenichiro Todoroki; Kenji Yamagata; Masatoshi Yamaguchi (88-93).
A liquid chromatographic (LC) method for sensitive and selective fluorometric determination of p-hydroxyphenylethylamino group containing compounds is described. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butanoyl chloride, followed by reversed-phase LC. The analytes, containing an amino moiety and a phenolic hydroxyl moiety in a molecule, were converted to the corresponding dipyrene-labeled derivatives by one-step derivatization. The dipyrene-labeled derivatives afforded intramolecular excimer fluorescence (440–540 nm), which can clearly be discriminated from the normal fluorescence (360–420 nm) emitted from reagent blanks. The derivatives of tyrosine and tyramine could be separated by reversed-phase LC on ODS column under conditions of isocratic elution. The detection limits (signal-to-noise ratio = 3) for tyrosine and tyramine were 4.5 and 2.6 fmol per 20 μL injection, which corresponded to analyte concentrations of 0.9 and 0.5 nM, respectively.
Keywords: p-Hydroxyphenylethylamino group containing compounds; Tyrosine; Tyramine; Liquid chromatography; Derivatization; Urine; Excimer fluorescence;

Simultaneous profiling analysis of urinary amino acids (AAs) and carboxylic acids (CAs) was combined with retention index (I) analysis for graphic recognition of abnormal metabolic state. The temperature-programmed I values of the AA and CA standards measured as ethoxycarbonyl (EOC)/methoxime (MO)/tert-butyldimethylsilyl (TBDMS) derivatives were used as the reference I values. Urine samples were subjected to the sequential EOC, MO and TBDMS reactions for the analysis by gas chromatography (GC) and GC–mass spectrometry. The complex GC profiles were then transformed into their respective I patterns in bar graphic forms by plotting the normalized peak area ratios (%) of the identified AAs and CAs against their reference I values as the identification numbers. When the present method was applied to infant urine specimens from normal controls and patients with inherited metabolic diseases such as phenylketonuria, maple syrup urine disease, methylmalonic aciduria or isovaleric aciduria, each I pattern of bar graph more distinctly displayed quantitative abundances of urinary AAs and CAs in qualitative I scale, thus allowing graphic discrimination between normal and abnormal states.
Keywords: Retention index analysis; Urinary amino acids and carboxylic acids; Ethoxycarbonylation; Methoximation; tert-Butyldimethylsilylation; Inherited metabolic disorders;

Interference free and simplyfied liquid chromatography-based determination of thiopurine S-methyltransferase activity in erythrocytes by Maurice N. Khalil; Norbert Erb; Philipe N. Khalil; Gabriele Escherich; Gritta E. Janka-Schaub (105-111).
The determination of the thiopurine S-methyltransferase activity (TPMT; EC has become an important issue during thiopurine therapy due to its known genetic polymorphism resulting in a wide range of TPMT activity. Therefore, the standard thiopurine drug regimen is associated with increased hematopoetic toxicity in patients with low or absent TPMT activity, whereas patients with high activity may be insufficiently treated. However, presently available methods are labour intensive and time consuming and tend towards too high or too low enzyme activity due to their methodological approach. The use of instable substrate solutions (6-MP or 6-TG), organic solvents like dimethyl sulfoxide and too high substrate and co-substrate saturation concentrations contribute to this phenomenon. We therefore, established an optimized and fast isocratic HPLC linked TPMT assay based on the enzymatic methylation of mercaptopurine or thioguanine in RBC lysates with S-adenosyl-l-methionine as methyl donor. Unspecific non-enzymatic methylation was not detectable. The recovery of 6-methyl-mercaptopurine was 97–102%, the intra- and interday variation between 1.0 and 5.0%, respectively. The assay dispenses with a time consuming extraction procedure with organic solvents, a heating step, and a gradient elution and is therefore, favourable for clinical routine application. The TPMT activity was measured in 62 untreated children with acute lymphoblastic leucemia at the time of diagnosis (activity = 34.0 ± 10.6 nmol/g Hb/h, range: 11.5–55.4 nmol/g Hb/h) and in 12 adult healthy volunteers (62.8 ± 7.7 nmol/g Hb/h, range: 48–82 nmol/g Hb/h) reflecting the wide measurable TPMT activity found in erythrocytes.
Keywords: Leukemia; Enzymes; Thiopourine S-methyltransferase; 6-Mercaptopurine; 6-Thioguanine;

A novel affinity sorbent system for direct bilirubin removal from human plasma was developed. These new adsorbents comprise Cibacron Blue F3GA as the specific ligand, and microporous membranous poly(tetrafluoroethylene) capillary (modified by coating with a hydrophilic layer of poly(vinyl alcohol) after activation) as the carrier matrix. The affinity adsorbents carrying 126.5 μmol Cibacron Blue F3GA/g polymer was then used to remove bilirubin in a flow-injection system. Non-specific adsorption on the poly(vinyl alcohol) coated capillary remains low, and higher affinity adsorption capacity, of up to 76.2 mg/g polymer was obtained after dye immobilization. The bilirubin adsorption capacity of the affinity capillary decreased with increase in the recirculation rate of plasma. The adsorption capacity increased with increase the temperature while decreased with increase the ionic strength. The maximum adsorption was only observed in neutral solution (pH 6–7). The adsorption isotherm fitted the Langmuir model well. These new adsorbents have higher velocity of mass transfer, better adsorption capacity, less fouling, longer service life and good reusability. The results of blood tests suggested the dye affinity capillary has good blood compatibility.
Keywords: Bilirubin removal; Cibacron Blue F3GA; Microporous membranous capillary; Bilirubin; Blood compatibility;