Journal of Chromatography B (v.798, #1)
News Section (N1-N2).
FMiii: Full-title Page (iii).
Characterization of human transferrin glycoforms by capillary electrophoresis and electrospray ionization mass spectrometry by V Sanz-Nebot; P González; I Toro; A Ribes; J Barbosa (1-7).
Carbohydrate Deficient Glycoprotein Syndrome (CDGS) is an inherited metabolic disease affecting all parts of the body. The biochemical diagnosis of this syndrome is based on the presence of a special marker in blood, Carbohydrate Deficient Transferrin (CDT), which is also a marker of chronic alcohol abuse. CDT is characterized by abnormal glycoforms of serum transferrin (Tf). In the present study, electrophoretic separation of human serum transferrin glycoforms was carried out using a bare fused-silica capillary and the glycoforms present in commercial Tf were baseline separated. The limit of detection (LOD) of human Tf was around the nmol concentration range. The LOD of the trisialo- and disialo-Tf, expressed as percentages of the tetrasialo-Tf peak area, were 0.5% for trisialo-Tf and 0.4% for disialo-Tf, and these values were appropriate for CDGS diagnosis. Moreover, Tf glycoforms were characterized using mass spectrometry (MS). The method was applied to the analysis of normal and pathological serum samples, after dilution. The results obtained suggest a way of making a rapid and simple CDGS diagnosis.
Keywords: Carbohydrate deficient glycoprotein syndrome; Glycoforms; Transferrin;
Fully automated on-line quantification of quetiapine in human serum by solid phase extraction and liquid chromatography by J Hasselstrøm; K Linnet (9-16).
A quantitative method for determination of quetiapine (QTP) in human serum is presented. The method is fully automated and based on high performance liquid chromatography (HPLC) with on-line solid phase extraction (SPE). The extraction procedure is based on a C2 cartridge, which is eluted with methanol. The eluate is injected onto a silica column with a mobile phase consisting of methanol:20 mM NH4CH3COO, pH 5.0 (99:1). Quetiapine is quantified by ultra-violet (UV) absorbance at 257 nM with trifluoperazine as the internal standard (I.S.). The extraction recoveries for quetiapine and trifluoperazine were 69 and 57%, respectively. The total inter day coefficient of variation was 11.1, 3.8 and 3.1% at 20, 500 and 1000 nM, respectively. The detection limit was 10.3 nM quetiapine. The method has been used in our therapeutic drug monitoring (TDM) laboratory where co-administered drugs often are observed. In an investigation of analytical interference from co-administered drugs, demethyl-mianserine was the only drug which interfered with the internal standard. There was no interference with quetiapine itself. The method showed good agreement with mass spectrometric quantification of quetiapine.
Size-exclusion flow extraction of bisphenol A in human urine for liquid chromatography–mass spectrometry by Koichi Inoue; Migaku Kawaguchi; Yukari Funakoshi; Hiroyuki Nakazawa (17-23).
We report an approach for assessing human exposure to bisphenol A (BPA), which involves measuring the glucuronide in urine sample that were subjected to a novel size-exclusion flow extraction method. The present approach includes the addition of 13 C 12 -BPA, enzymatic deconjugation, and the proposed sample preparation method. The sample solution is separated and detected by liquid chromatography–mass spectrometry (LC–MS). The following are used for the LC–MS: a reversed-phase separation column, electrospray ionization (ESI), negative mode, and single ion monitoring (SIM) with m/z 227 for BPA and m/z 239 for 13 C 12 -BPA. The detection limit was 0.1 ng ml−1 and the calibration curves (0.45–90 ng ml−1) had correlation coefficients exceeding 0.999. To urine samples requiring deglucuronidation, β-glucuronidase was added followed by incubation at 37 °C for 3 h. After the enzymatic treatment, the samples were subjected to the extraction in the reversed-phase (ODS) and size-exclusion (GPC) modes. It was possible to extract, clean up and concentrate BPA in a single run of 20 min by means of the novel extraction method. The method enables the determination of standards and may be applied to the detection of trace amounts of BPA in human urine samples.
Keywords: Sample preparation; Bisphenol A;
Study of the serum albumin-polyethyleneglycol interaction to predict the protein partitioning in aqueous two-phase systems by Beatriz Farruggia; Bibiana Nerli; Guillermo Picó (25-33).
The theoretical framework based only on the excluded volume forces is not enough to explain the bovine serum albumin partitioning behaviour in aqueous biphasic systems. The goal of this work is to look at the phase separation via the polymer effect on the water structure. Our findings suggest that polyethyleneglycol 600-protein interaction is conducted by van der Waals forces between the hydrophobic surfaces from PEG and protein molecules, which implies the rupture of hydrogen bonds from the structured water in their neighbours. Therefore, the protein will concentrate in the most water-structured phase (polyethyleneglycol) in order to reach the minimal free energy condition. When polyethyleneglycol molecular weight increases, its exclusion from protein surface prevails, thus pushing the bovine serum albumin to the bottom phase.
Keywords: Protein partitioning; Aqueous two-phase systems; Albumin; Polyethyleneglycol;
Application of ultrafiltration method to measurement of catecholamines in plasma of human and rodents by high-performance liquid chromatography by Jun Ueyama; Kiyoyuki Kitaichi; Mitsunori Iwase; Kenji Takagi; Kenzo Takagi; Takaaki Hasegawa (35-41).
In order to develop a reliable, simple and routine method using small sample volume to determine norepinephrine (NE) and epinephrine (E) concentrations in plasma of humans and rodents, we utilize the ultrafiltration (UF) method by Ultrafree-MC filter device and a high-performance liquid chromatography equipped with electrochemical detector (HPLC-ECD) to detect NE and E. Optimum UF and HPLC conditions were as follows: the filter nominal molecular weight limit size is 30,000, the pH of added phosphate buffer to each plasma sample for UF is 3.0, and the mobile phase is 0.1 M phosphate buffer (pH 3)/acetonitrile (98:2) containing 0.05% sodium disulfite and 0.001% EDTA 2Na. The plasma samples and 1.0 M phosphate buffer (pH 3) containing 3,4-dihydroxybenzylamine (DHBA), as an internal standard, was mixed and poured into the UF units. After the centrifugation for 60 min at 13,000×g at 4 °C, the filtrate was directly injected into HPLC. The calibration curve of NE and E was linear for the concentrations studied (20–400 pg) with a correlation coefficient of >0.999. Intra-assay coefficients of variation for NE and E using this method were less than 3%. The method also correlated well with the well-established alumina method (r=0.954). The present findings suggest that a newly-developed UF method with HPLC-ECD would apply successfully to measure plasma NE and E concentrations in humans and rodents.
Keywords: Ultrafiltration; Catecholamines;
Determination of vertilmicin in rat serum by high-performance liquid chromatography using 1-fluoro-2,4-dinitrobenzene derivatization by Maojin Zhou; Guangli Wei; Youping Liu; Yuming Sun; Shuhua Xiao; Long Lu; Changxiao Liu; Dafang Zhong (43-48).
A procedure for the high-performance liquid chromatographic determination of vertilmicin in rat serum was described using pre-column derivatization. The serum proteins were precipitated with acetonitrile and vertilmicin in the supernatant was derivatized with 1-fluoro-2,4-dinitrobenzene. Etimicin was selected as the internal standard. The mobile phase consisted of methanol−20 mM ammonium acetate (80:20, v/v), and flow-rate was 0.9 ml/min. Ultraviolet detection was set at 365 nm. The reaction products were chromatographed on a C18 column kept at 40 °C. A good linearity was found in the range of 0.5–250 μg/ml. Both intra- and inter-day precisions of vertilmicin, expressed as the relative standard deviation, were less than 7.4%. Accuracy, expressed as the relative error, ranged from −0.1 to 3.6%. The mean absolute recovery of vertilmicin at three different concentrations was 92.5%. Serum volumes of 50 μl were sufficient for the determination of vertilmicin. The method was proved suitable for the pharmacokinetic study of vertilmicin in rats.
Keywords: Derivatization, LC; Vertilmicin; 1-Fluoro-2,4-dinitrobenzene;
Liquid chromatographic determination of ceterizine hydrochloride and paracetamol in human plasma and pharmaceutical formulations by Basavaraj S Nagaralli; Jaldappa Seetharamappa; Babu G Gowda; Mahaveer B Melwanki (49-54).
An accurate, simple, reproducible and sensitive HPLC method for the determination of ceterizine hydrochloride (CTZH) and paracetamol (PARA) has been developed and validated. The separation of CTZH, PARA and Nimesulide (the internal standard) was achieved on a CLC C18 (5 μ, 25 cm×4.6 mm i.d) column using UV detection at 230 nm. The mobile phase was consisted of acetonitrile–water (55:45 v/v). The linear ranges of detection for CTZH and PARA were found to be 0.715–55 μg/ml (r 2=0.9985) and 0.55–39 μg/ml (r 2=0.9957) respectively. Intra- and inter-day assay relative standard deviations were less than 1%. The method has been applied successfully to the determination of binary combination of CTZH and PARA in human plasma and pharmaceutical preparations. There was no interference from drugs commonly administered with CTZH and PARA. The method has been shown to be linear, reproducible, specific, and rugged.
Keywords: Ceterizine hydrochloride; Paracetamol;
Liquid chromatographic–mass spectrometric determination of the metabolism and disposition of the anti-retroviral nucleoside analogs zidovudine and lamivudine in C57BL/6N and B6C3F1 mice by Lee D Williams; Linda S Von Tungeln; Frederick A Beland; Daniel R Doerge (55-62).
Transmission of HIV from mother to infant can be effectively prevented by zidovudine (3′-azido-3′-deoxythymidine; AZT) alone or in combination with other anti-retroviral drugs; however, significant evidence for genotoxicity, including transplacental carcinogenicity in mice, has been reported for AZT. A method, based upon solid phase extraction (SPE) in the 96-well format, gradient liquid chromatography (LC), and electrospray mass spectrometry (MS), was developed and validated to measure serum concentrations in maternal C57BL/6N and fetal B6C3F1 mice of the nucleoside analogs AZT, lamivudine ((-)2′,3′-dideoxy-3′-thiacytidine; 3TC), and several metabolites selected based on importance in detoxification and bioactivation reactions. After intravenous (IV) and oral dosing with either 400 mg/kg AZT or 200 mg/kg 3TC, pharmacokinetics were determined for AZT, AZT-5′-glucuronide, 3′-amino-3′-deoxythymidine (AMT), AZT-5′-phosphate, 3TC, and 3TC-5′-phosphate in serum of adult female mice. Pharmacokinetics were also determined in spleen for AZT-5′-phosphate and 3TC-5′-phosphate following IV dosing. In addition, a preliminary assessment was made of placental transfer of AZT and 3TC and the presence of metabolites in the fetal compartment. The method described provides a means to evaluate thoroughly metabolism and disposition of anti-retroviral nucleoside analogs in maternal and fetal mice for comprehensive studies of genotoxicity.
Keywords: Zidovudine; Lamivudine;
Bioanalysis of zosuquidar trihydrochloride (LY335979) in small volumes of human and murine plasma by ion−pairing reversed-phase high-performance liquid chromatography by E.M Kemper; M Ouwehand; J.H Beijnen; O van Tellingen (63-68).
We have developed and validated a sensitive and selective method for the quantitative determination of the P-glycoprotein inhibitor zosuquidar (LY335979) in human and murine plasma using only 50 μl sample volumes. Sample pretreatment involved liquid-liquid extraction with tert-butyl methyl ether. Zosuquidar and the internal standard chlorpromazine were separated using a narrow bore column (2.1 mm×150 mm) packed with 3.5 μm symmetry C18 material. The mobile phase consisted of 38% (v/v) acetonitrile in 50 mM ammonium acetate buffer pH 3.8 containing 0.005 M 1-octyl sulfonic acid and was delivered at 0.2 ml/min. Detection was performed with a fluorescence detector set at an excitation wavelength of 260 nm and an emission wavelength of 460 nm. The calibration curve was prepared in blank human plasma and was linear over the dynamic range (10–1000 ng/ml). The lower limit of quantitation was 20 ng/ml. The validation results showed that the assay was selective and reproducible. Within the range of the calibration curve the accuracy was close to 100% and within-day and between-day precision were within the generally accepted 15% range. This method was applied to study the pharmacokinetics of i.v. administered zosuquidar in mice. The sensitivity of the assay was sufficient to determine the drug concentration in plasma samples obtained up to 24 h after administration.
Keywords: Zosuquidar trihydrochloride;
Optimization of the separation of a complex mixture of natural and synthetic anabolic steroids by micellar liquid chromatography by R Izquierdo-Hornillos; R Gonzalo-Lumbreras (69-77).
A systematic optimization of the HPLC separation of a complex mixture containing natural and synthetic anabolic steroids by micellar liquid chromatography using a Hypersil (150 mm×3.0 mm i.d., 5 μm) C18 column and UV detection at 245 nm (exception is made for oxymetolone and danazol which were monitorized at 280 nm) has been carried out. The isocratic micellar mobile phases (from binary to quaternary) consisted of sodium dodecyl sulphate and organic modifiers such as acetonitrile, tetrahydrofuran, propanol, butanol or pentanol. The effect of the organic modifiers, surfactant concentration, temperature, ionic strength and flow-rate on the separation has been studied. A micellar mobile phase 5% propanol and 40 mM surfactant allowed the separation of 12 steroids out of 14 tested in about 20 min. A bivariant optimization method for the micellar mobile phase propanol-surfactant corroborated the above results.
Keywords: Anabolic steroids;
Determination of paclitaxel in mouse plasma and brain tissue by liquid chromatography–mass spectrometry by Ping Guo; Jianguo Ma; Shaolan Li; James M. Gallo (79-86).
Paclitaxel is an anticancer agent extracted from the bark of the yew tree and is widely used in chemotherapy for solid tumors, including non-small cell lung cancer and ovarian carcinoma. Most assays to measure paclitaxel in plasma require a large amount of sample (0.4–1 ml) to achieve the necessary sensitivity, and are not suitable when only small sample sizes are available. To circumvent this latter limitation, we developed a sensitive liquid chromatography–mass spectrometry (LC–MS) method for the determination of paclitaxel in plasma based on the use of small sample volumes (50 μl plasma). A solid phase extraction procedure was employed that enabled the eluent to be directly injected onto a reversed phase chromatographic HPLC system using positive electrospray ionization followed by mass spectrometric detection. The extraction recoveries of paclitaxel were 98 and 83% from plasma and brain tissues, respectively. The mobile phase consisted of 50% acetonitrile in 0.1% formic acid that was pumped at 0.2 ml/min to yield a retention time for paclitaxel of 6.2 and 5.4 min for cephalomannine, the internal standard. The method has been validated at paclitaxel plasma concentrations from 0.036 to 9.9 μg/ml, and from 0.054 to 1.96 μg/ml in brain homogenates. A sensitive and specific assay for paclitaxel has been developed that has the advantages of using small sample sizes, and a single extraction step without solvent evaporation.
Quantitative determination of circulating and urinary asymmetric dimethylarginine (ADMA) in humans by gas chromatography–tandem mass spectrometry as methyl ester tri(N-pentafluoropropionyl) derivative by Dimitrios Tsikas; Bibiana Schubert; Frank-Mathias Gutzki; Jörg Sandmann; Jürgen C Frölich (87-99).
Asymmetric dimethylarginine (ADMA; N G,N G-dimethyl-l-arginine) is the most important endogenous inhibitor of nitric oxide synthase and a potential risk factor for cardiovascular diseases. This article describes a gas chromatographic–tandem mass spectrometric (GC–tandem MS) method for the accurate quantification of ADMA in human plasma or serum and urine using de novo synthesized [ 2 H 3 ]-methyl ester ADMA (d3Me-ADMA) as the internal standard. Aliquots (100 μl) of plasma/serum ultrafiltrate or native urine and of aqueous solutions of synthetic ADMA (1 μM for plasma and serum; 20 μM for urine) are evaporated to dryness. The residue from plasma/serum ultrafiltrate or urine is treated with a 100 μl aliquot of 2 M HCl in methanol, whereas the residue of the ADMA solution is treated with a 100 μl aliquot of 2 M HCl in tetradeuterated methanol. Methyl esters are prepared by heating for 60 min at 80 °C. After cooling to room temperature, the plasma or urine sample is combined with the d3Me-ADMA sample, the mixture is evaporated to dryness, the residue treated with a solution of pentafluoropropionic (PFP) anhydride in ethyl acetate (1:4, v/v) and the sample is incubated for 30 min at 65 °C. Solvent and reagents are evaporated under a stream of nitrogen gas, the residue is treated with a 200 μl aliquot of 0.4 M borate buffer, pH 8.5, and toluene (0.2 ml for plasma, 1 ml for urine). Reaction products are extracted by vortexing for 1 min, the toluene phase is decanted, and a 1 μl aliquot is injected into the GC–tandem MS instrument. Quantitation is performed by selected reaction monitoring (SRM) of the common product ion at m/z 378 which is produced by collision-induced dissociation of the ions at m/z 634 for endogenous ADMA and m/z 637 for d3Me-ADMA. In plasma and urine of healthy humans ADMA was measured at concentrations of 0.39±0.06 μM (n=12) and 3.4±1.1 μmol/mmol creatinine (n=9), respectively. The limits of detection and quantitation of the method are approximately 10 amol and 320 pM of d3Me-ADMA, respectively.
Keywords: Derivatization; Asymmetric dimethylarginine;
Liquid chromatography method for plant and mammalian lignans in human urine by Tarja Nurmi; Sari Voutilainen; Kristiina Nyyssönen; Herman Adlercreutz; Jukka T Salonen (101-110).
Recently new mammalian lignan precursors were identified but no analysis methods are available for assay of those compounds in human urine. Previously published methods were developed for GC–MS about only two plant lignans were included. Consequently, a method for HPLC equipped with a coulometric electrode array detector was developed to measure plant and mammalian lignans in human urine. The plant lignans, secoisolariciresinol (Seco), matairesinol (Mat), lariciresinol (Lar), pinoresinol (Pin), syringaresinol (Syr) and isolariciresinol (IsoL) were included into the new method together with two mammalian lignans, enterolactone (Enl) and enterodiol (End). Validation of the method demonstrated that it could be applied to normal urine containing low amounts of plant lignans and moderate amounts of mammalian lignans, but the method was also applicable for samples from study subjects in supplementation studies, i.e. sample with very high concentrations of mammalian lignans. The method was found to be a useful tool for studies on plant lignan intake and the activity of micro flora in the metabolism of plant lignans.
Keywords: Lignans; Enterolactone;
Determination of thiopurine S-methyltransferase activity in erythrocytes using 6-thioguanine as substrate and a non-extraction liquid chromatographic technique by Loretta Theresa Ford; Jonathan David Berg (111-115).
A non-extraction high-performance liquid chromatographic (HPLC) method has been developed for the determination of 6-methylthioguanine (6-MTG), as part of the determination of thiopurine S-methyltransferase activity (TPMT) in erythrocytes. Erythrocyte lysate is added to a glass vial containing substrates and incubation buffer, which is then sealed for the rest of the analysis. Enzyme incubation, sample preparation, and analysis are then undertaken without further sample-handling steps. The need for a solvent extraction step has been overcome by heating the incubate to 85 °C to stop the enzyme reaction. The heat inactivation step precipitates protein which upon centrifugation forms a thin film in the bottom of the glass vial enabling the supernatant to be injected directly onto the HPLC system. The assay shows excellent precision and recovery with a within-batch imprecision giving a co-efficient of variation of 2.9% (mean=41.5 nmol 6-MTG/g Hb/h, n=10) and 5.1% (mean=12.6 nmol 6-MTG/g Hb/h, n=10). The between-batch imprecision gives a co-efficient of variation of 8.2% (mean=11.1 nmol 6-MTG/g Hb/h, n=11) and 7.3% (mean=41.0 nmol 6-MTG/g Hb/h, n=16). Determination of the TPMT activity in 120 people shows a range of enzyme activity of 11.3–63.8 nmol 6-MTG/g Hb/h with a mean and median activity of 34.8 and 34.2 nmol 6-MTG/g Hb/h, respectively. TPMT is increasingly used in clinical practice to ensure optimisation of treatment with thioguanine drugs. This direct HPLC method minimises sample-handling, reduces inherent imprecision, the possibility of laboratory error and with the potential for further automation, makes it ideal for use in a regional referral laboratory.
Keywords: Enzymes; Thiopurine S-methyltransferase; 6-Thioguanine;
Quantitative chromatographic determination of several benzimidazole anthelmintic molecules in parasite material by Lourdes Mottier; Luis Alvarez; Carlos Lanusse (117-125).
A specific, precise and accurate high-performance liquid chromatographic (HPLC) analytical method has been developed for the quantitative determination of different benzimidazole (BZD) anthelmintics in parasite material (Moniezia benedeni). Mebendazole (MBZ), oxibendazole (OBZ), flubendazole (FLBZ), albendazole (ABZ) ricobendazole (RBZ), albendazole sulphone (ABZSO2), fenbendazole (FBZ), oxfendazole (OFZ) and fenbendazole sulphone (FBZSO2) were measured simultaneously in M. benedeni, a sheep and cattle cestode parasite used as a model of the biological matrix. The recovery, linearity, precision, accuracy, limits of detection and quantification of the method were determined. Drug extraction from the parasite’s tissue homogenate was performed using methanol (liquid phase extraction), and after solvent evaporation, the residual material was cleaned up by solid phase extraction prior to analysis by reversed-phase HPLC. The resolution of all the BZD molecules assayed was obtained on a C18 reversed-phase (5 μm, 250 mm×4.6 mm) column using acetonitrile and ammonium acetate as the mobile phase and ultraviolet (UV) detection at 292 nm. Regression analyses for all the BZD compounds assayed were linear at concentrations ranging from 1.61 to 64.21 nmol/100 mg protein (triplicate determinations) showing correlation coefficients greater than 0.9922. The developed method is efficient for the simultaneous determination of several benzimidazole anthelmintic molecules in parasite material and useful for the ex vivo and in vivo characterisation of the kinetics of drug uptake/diffusion in target parasites, which seems to be relevant to optimise parasite control both in human and veterinary medicine.
Keywords: Benzimidazole anthelmintics;
Three-phase liquid-phase microextraction of weakly basic drugs from whole blood by Hege Grefslie Ugland; Mette Krogh; Léon Reubsaet (127-135).
The behaviour of weak basic analytes in liquid-phase microextraction (LPME) and the optimisation of parameters in whole blood are described. Benzodiazepines and non-benzodiazepine drugs were chosen as model substances. Liquid-phase microextraction based on disposable polypropylene hollow fibres was used in the three-phase extraction of five weak bases from whole blood. The sample work up with the liquid-phase microextraction technique can be impeded by low recovery due to incomplete trapping in the acceptor phase of weakly basic drugs and the complexity of the whole blood matrix. Different parameters related to this problem were experimentally studied. Additionally the stability of the analytes was examined because of low pH in the acceptor phase. The investigation resulted in optimised LPME conditions for the extraction of weak bases from whole blood. The parameters limiting the recovery were evaluated.
Keywords: Three-phase liquid microextraction; Weak bases;
Determination of fluoroquinolones in human urine by liquid chromatography coupled to pneumatically assisted electrospray ionization mass spectrometry by O Ballesteros; I Toro; V Sanz-Nebot; A Navalón; J.L Vı́lchez; J Barbosa (137-144).
The fluoroquinolones are synthetic antimicrobial agents widely used in human and veterinary medicine. The aim of this work was to develop a method of characterization and determination of three widely used fluoroquinolones (norfloxacin, ciprofloxacin and ofloxacin) in human urine by liquid chromatography (LC) coupled to pneumatically assisted electrospray ionization (ESI) mass spectrometry (MS). For this purpose, the operational parameters of the electrospray interface were optimized in order to obtain the best signal stability and the highest sensitivity of the fluoroquinolones. The three fluoroquinolones studied and enrofloxacin, used as internal standard, were extracted from human urine samples by solid-phase extraction (SPE) and the previously established LC-UV method was successfully coupled with the MS system. The mass spectra obtained provide adequate information for identification purposes. Quality parameters were determined and satisfactory results were obtained. Likewise, the method detection limit was about 10 ng ml−1 for the three fluoroquinolones studied employing selected-ion monitoring mode.
Validated method for the simultaneous determination of Δ9-tetrahydrocannabinol (THC), 11-hydroxy-THC and 11-nor-9-carboxy-THC in human plasma using solid phase extraction and gas chromatography–mass spectrometry with positive chemical ionization by Richard A Gustafson; Eric T Moolchan; Allan Barnes; Barry Levine; Marilyn A Huestis (145-154).
A fully validated, highly sensitive and specific method for the extraction and quantification of Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THCCOOH) in plasma is presented. This method incorporates Escherichia coli β-glucuronidase hydrolysis to cleave glucuronic acid moieties to capture total analyte concentrations, and simultaneous solid phase extraction (SPE) of the three analytes in a single eluant with separation and quantification on a bench-top positive chemical ionization (PCI) gas chromatography–mass spectrometry (GC–MS) in the selected ion monitoring (SIM) mode. Quantitation was achieved by the addition of deuterated analogues for each analyte as internal standards (IS). Limits of quantitation (LOQ) were 0.5, 0.5 and 1.0 for THC, 11-OH-THC and THCCOOH, respectively, with linearity ranging up to 50 ng/ml for THC and 11-OH-THC, and 100 ng/ml for THCCOOH. Absolute recoveries ranged from 67.3 to 83.5% for all three analytes. Intra-assay accuracy and precision ranged from 1.2 to 12.2 and 1.4 to 4.7%, respectively. Inter-assay accuracy and precision ranged from 1.4 to 12.2 and 3.1 to 7.3%, respectively. This method was used to analyze plasma samples collected from individuals participating in a controlled oral THC administration study. Statistically significant (P≤0.05) increases of 40% for 11-OH-THC and 42% for THCCOOH concentrations were found between hydrolyzed and non-hydrolyzed results. This method will be utilized in ongoing controlled cannabinoid administration studies and may be a useful analytical procedure for the fields of forensic toxicology and cannabinoid pharmacology.
Keywords: Δ9-Tetrahydrocannabinol; 11-Hydroxy-Δ9-tetrahydrocannabinol; 11-Nor-9-carboxy-Δ9-tetrahydrocannabinol;
Microdialysis for the determination of acetaldehyde and ethanol concentrations in the striatum of freely moving rats by Mostofa Jamal; Kiyoshi Ameno; Mitsuru Kumihashi; Setsuko Ameno; Takako Kubota; Weihuan Wang; Iwao Ijiri (155-158).
The subject of the present paper is the simultaneous determination of ethanol (EtOH) and acetaldehyde (AcH) concentrations in the striatum of freely moving rats using an in vivo microdialysis followed by head-space gas chromatography (GC). Major operation conditions of GC were as follows: column, injector and detector temperatures 90, 110 and 200 °C, respectively; Supelcowax™ wide bore capillary column (60 m length, 0.53 mm i.d., 2 μm film thickness); carrier gas, nitrogen; flow rate, 20 ml/min. The recovery of EtOH and AcH at a concentration 40 mM and 250 μM, respectively, by microdialysis showed a maximum of 83.8±12.2 and 51.2±6.5%, respectively, at a flow rate of 0.8 μl/min. A good linear calibration curve in the concentration range of 5–50 mM for EtOH (r=0.998), and 10–250 μM for AcH (r=0.988) was observed. Microdialysates were collected for 10 min each after insertion of probe into the striatum. Rats were treated with cyanamide (100 mg/kg, a potent aldehyde dehydrogenase inhibitor) and 60 min later with EtOH (1 g/kg) intraperitoneally. A 10 min sample was about 8 μl. This volume was mixed with 40 μl of 0.002% t-butanol as an internal standard in 0.6N perchloric acid, and then analyzed by head-space GC method. The peak EtOH and AcH concentrations in the striatal dialysates reached maximum at 30 min, and then gradually decreased. This method represents a reasonable tool to quantify in vivo both AcH and EtOH levels simultaneously in rat brain.
Keywords: Microdialysis; Acetaldehyde; Ethanol;
Measurement of plasma pristanic, phytanic and very long chain fatty acids by liquid chromatography-electrospray tandem mass spectrometry for the diagnosis of peroxisomal disorders by David W Johnson; Minh-Uyen Trinh; Tomoyuki Oe (159-162).
High pressure liquid chromatography with a narrow bore C8 column has been used to separate pristanic, phytanic and very long chain fatty acids, important in the diagnosis of peroxisomal disorders, for their accurate isotope dilution quantification by tandem mass spectrometry. The fatty acids, isolated from plasma, were analysed as trimethylaminoethyl ester (quaternary ammonium) derivatives. Analysis time was 2.5 h and sample requirement was 10 μl of plasma. Good agreement with GC–MS methods for the levels of pristanic and phytanic acids, C26:0/C22:0 and C24:0/C22:0 ratios were obtained for 12 plasma samples from peroxisomal disorder patients and a set of controls.
Keywords: Pristanic acid; Phytanic acid; Very long chain fatty acids;
Detection of 3-methoxy-4-hydroxyphenylglycol in rabbit skeletal muscle microdialysate by Noriyuki Tokunaga; Toji Yamazaki; Tsuyoshi Akiyama; Hidezo Mori (163-166).
A high-performance liquid chromatography with electrochemical detection (HPLC–ED) method is described for determination of 3-methoxy-4-hydroxyphenylglycol (MHPG) in microdialysate from the skeletal muscle interstitial space. Using a microdialysis technique, we sampled 30 μl dialysate from the skeletal muscle interstitial space and injected dialysate directly into HPLC–ED system. The control MHPG concentration of dialysate was 213±18 pg/ml. The MHPG concentrations were reduced by entacapone (catechol-O-methyltransferase inhibitor, COMT), augmented by local infusion of dihydroxyphenylglycol. This system offers a new possibility for simple, rapid monitoring of MHPG as an index of COMT activity in skeletal muscle.
Determination of gatifloxacin in human serum and urine by high-performance liquid chromatography with ultraviolet detection by Brian R. Overholser; Michael B. Kays; Kevin M. Sowinski (167-173).
A simple and sensitive HPLC method for the determination of gatifloxacin concentrations in human serum and urine was developed and validated. Serum proteins were removed by ultrafiltration through a filtering device after adding a displacing agent. Urine samples were diluted with mobile phase prior to injection. Separation was achieved with a C18 reverse-phase column and gatifloxacin concentrations were determined using ultraviolet detection. The quantitation limits of the assay were 100 ng/ml in serum and 1.0 μg/ml in urine. The assay method was successfully applied to a pharmacokinetic study of gatifloxacin in healthy volunteers.
Determination of lactate dehydrogenase in human erythrocytes by capillary electrophoresis with electrochemical detection by Wei Wang; Xuemei Sun; Wenrui Jin (175-178).
Capillary electrophoresis (CE) was employed to analyze lactate dehydrogenase (LDH) in human erythrocytes using an amperometric detector with a carbon fiber micro-disk bundle electrode. LDH activity was measured by determining the amount of NADH generated by LDH through a enzyme-catalyzed reaction between NAD+ and lithium lactate. The factors influencing the enzyme-catalyzed reaction, separation and detection were examined and optimized. The following conditions were suitable for the determination of LDH: running buffer, 5.0×10−2 mol/l Tris–HCl (pH 7.5); separation voltage, 20.0 kV; detection potential, 1.00 V (versus saturated calomel electrode (SCE)). The conditions of enzyme-catalyzed reaction were: reaction buffer, 5.0×10−2 mol/l Tris–HCl (pH 9.3); substrates, 5.0×10−2 mol/l lithium lactate and 5.0×10−3 mol/l NAD+; reaction time, 10 min. The concentration limit of detection (LOD) of the method was 0.017 U/ml at a signal-to-noise (S/N) ratio of 3, which corresponded to 1.10×10−10 mol/l, and the mass LOD was 2×10−20 mol. The linear dynamic range was 0.039–4.65 U/ml for the injection voltage of 5.0 kV and injection time of 10 s. The relative standard deviation (R.S.D.) was 0.85% for the migration time and 1.8% for the electrophoretic peak area. The method was applied to determine LDH in human erythrocytes. The recovery of the method was between 98 and 101%.
Keywords: Electrochemical detection; Lactate dehydrogenase; Enzymes;