Journal of Chromatography B (v.845, #1)
Editorial Board (iii).
Review of methodology for the determination of benzimidazole residues in biological matrices by Martin Danaher; Hendrik De Ruyck; Steven R.H. Crooks; Geraldine Dowling; Michael O’Keeffe (1-37).
Benzimidazoles are anthelmintic agents widely used in the treatment of parasitic infections in a range of species and as fungicidal agents in the control of spoilage of crops during storage and transport. In this paper, the more important benzimidazoles are introduced and their pharmacological effects and physiochemical properties discussed. The metabolism of these drugs is described relating to the occurrence and persistence of residues in biological matrices, providing information for selection of suitable matrices and target residues for testing. Methods for determination of benzimidazoles are reviewed for a range of biological matrices. The importance of selecting suitable extraction and clean-up procedures is discussed, along with the difficulties encountered in adapting single residue methods to multi-residue methods. The importance of suitable detection systems for determination of benzimidazoles, namely, screening, HPLC, GC and confirmatory methods is described in detail. The future for benzimidazole residue analysis is discussed, focusing on selection of appropriate residues for screening methods and protocols for confirmation of benzimidazole residues.
Keywords: Benzimidazole residues; Animal tissues; SPE; HPLC; LC–MS/MS;
Aqueous two-phase extraction for protein recovery from corn extracts by Zhengrong Gu; Charles E. Glatz (38-50).
Corn has been used as an expression host for several recombinant proteins with potential for large-scale production. Cost-effective downstream initial recovery, separation and concentration remain a challenge. Aqueous two-phase (ATP) partitioning has been used to recover and concentrate proteins from fermentation broths and offers advantages for integration of those steps with biomass removal. To examine the applicability of ATP partitioning to recombinant protein purification from corn endosperm and germ, ATP system parameters including poly(ethylene glycol) (PEG) molecular weight (MW), phase-forming salt, tie line length (TLL), and pH were manipulated to control partitioning of extracted native proteins from each fraction. Moderate PEG MW, reduction of phase ratio, and added NaCl effected complete recovery of the hydrophobic model protein lysozyme in the top phase with ca. 5× enrichment and illustrates a favorable match of recombinant protein characteristics, expression host, and separation method. Furthermore, integration of protein extraction with the partitioning reduced the load of contaminating host proteins relative to the more traditional separate steps of extraction followed by partitioning. Performance of the integrated partitioning was hindered by endosperm solids loading, whereas for germ, which has ca. 35× higher aqueous soluble protein, the limit was protein solubility. For more hydrophilic model proteins (the model being cytochrome c), effective separation required further reduction of PEG MW to effect more partitioning of host proteins to the top phase and enrichment of the model protein in the lower phase. The combination of PEG MW of 1450 with 8.5 wt.% NaCl addition (Na2SO4 as the phase-forming salt) provided for complete recovery of cytochrome c in the lower phase with enrichment of 9× (germ) and 5× (endosperm). As a result of lower-phase recovery, the advantage of simultaneous removal of solids is lost. The lower solubility of native endosperm proteins results in higher purity for the same enrichment.
Keywords: Partitioning; Aqueous two-phase; Corn; Protein recovery; Extraction;
Simultaneous determination of 8 HIV protease inhibitors in human plasma by isocratic high-performance liquid chromatography with combined use of UV and fluorescence detection: Amprenavir, indinavir, atazanavir, ritonavir, lopinavir, saquinavir, nelfinavir and M8-nelfinavir metabolite by R. Verbesselt; E. Van Wijngaerden; J. de Hoon (51-60).
A simple, accurate and fast method was developed for determination of the commonly used HIV protease inhibitors (PIs) amprenavir, indinavir, atazanavir, ritonavir, lopinavir, nelfinavir, M8-nelfinavir metabolite and saquinavir in human plasma. Liquid–liquid extraction was used with hexane/ethylacetate from buffered plasma samples with a borate buffer pH 9.0. Isocratic chromatographic separation of all components was performed on an Allsphere hexyl HPLC column with combined UV and fluorescence detection. Calibration curves were constructed in the range of 0.025–10 mg/l. Accuracy and precision of the standards were all below 15% and the lowest limit of quantitation was 0.025 mg/l. Stability of quality control samples at different temperature conditions was found to be below 20% of nominal values. The advantages of this method are: (1) inclusion and determination of the newly approved atazanavir, (2) simultaneous isocratic HPLC separation of all compounds and (3) increased specificity and sensitivity for amprenavir by using fluorescence detection. This method can be used for therapeutic drug monitoring of all PIs currently commercialised and is now part of current clinical practice.
Keywords: Protease inhibitors; Drug monitoring; Chromatography;
Development of automated online gel permeation chromatography–gas chromatograph mass spectrometry for measuring multiresidual pesticides in agricultural products by Li-Bin Liu; Yuki Hashi; Ya-Ping Qin; Hai-Xia Zhou; Jin-Ming Lin (61-68).
An automated online gel permeation chromatography–gas chromatograph mass spectrometer (GPC–GC/MS) was developed for the rapid determination of residual pesticides in agricultural products. Pesticides were extracted from homogenized food samples with acetonitrile and decontaminated via the matrix solid-phase dispersion (MSPD) technique, using a primary secondary amine as sorbent prior to GPC–GC/MS analysis. A slightly modified preparation method and automated GPC step proved useful in minimizing matrix interference. To evaluate the performance of the system, 97 target pesticides were spiked at a concentration of 0.1 mg/kg into a range of food types, including potato, cabbage, carrot, apple, orange, cucumber, and rice. A low flow rate of 0.1 mL/min in GPC resulted in a 40-fold reduction in solvent consumption compared with conventional GPC column applications. The combination of MSPD technique and GPC–GC/MS for the analysis of the 97 pesticides can be accomplished within 90 min. Most pesticides were recovered in the range of 70–120%, with relative standard deviation generally less than 10%. The results demonstrate that the method can be successfully applied with acceptable recoveries to a broad range of target pesticides within a diverse range of food types.
Keywords: Residual pesticides; Agricultural products; GPC–GC/MS;
Determination of proteins in infant formula by high-performance liquid chromatography–electrospray tandem mass spectrometry by Soon Sun Hong; Jeong Hill Park; Sung Won Kwon (69-73).
To determine the protein content of formula, gel electrophoresis was performed on the infant formula samples and the entire protein patterns were analyzed by nano-high performance liquid chromatography–electrospray tandem mass spectrometry (nano-HPLC/ESI/MS/MS). From the commercial infant formula profiled in this study, a total of 154 peptides, corresponding to 31 unique proteins were identified by nano-HPLC/ESI/MS/MS. Each of the identified peptides was reconfirmed by a strict integrated approach using tandem mass spectra. This protein profiling method using gel electrophoresis coupled with nano-HPLC/ESI/MS/MS and manual evaluation is a sensitive and accurate method for protein identification as well as a powerful tool for monitoring various types of food products.
Keywords: Infant formula; High-performance liquid chromatography; Tandem mass spectrometry; Food analysis;
A confirmatory analysis of malachite green residues in rainbow trout with liquid chromatography–electrospray tandem mass spectrometry by Kirsi Halme; Erja Lindfors; Kimmo Peltonen (74-79).
A quantitative liquid chromatography–tandem mass spectrometric (LC–MS/MS) method has been developed for the determination of malachite green (MG) and its metabolite leucomalachite green (LMG) in fish. Residues were extracted with an acetonitrile–acetate buffer and purified using the automated solid-phase extraction (ASPEC). Residues were analyzed with a reversed-phase LC–MS/MS using a positive-ion electrospray ionisation (ESI). Isotope-labelled leucomalachite green (LMG-D5) was used as an internal standard for the quantification of LMG residues. The related dye, brilliant green (BG) was used as an instrumental standard. Identification and quantification of analytes were based on the ion transitions monitored by multiple reaction monitoring (MRM). The decision limit (CCα) for MG and LMG was 0.13 and 0.16 μg kg−1. The respective detection capabilities (CCβ) were 0.22 and 0.27 μg kg−1. The absolute recovery (repeatability SDr) was in the range of 58–65% (7.8–11.2%) for MG and 59–68% (9.7–16.9%) for LMG. LMG was quantified also based on the internal standard, giving a recovery (repeatability SDr) of 103–110% (4.8–9.3%). The method was further evaluated by analyzing a total of 34 fish residue monitoring samples, of which eight samples were found to be non-compliant containing low residues of LMG.
Keywords: Malachite green; Leucomalachite green; Fish; LC–MS/MS; Confirmation;
Assay for spermidine synthase activity by micellar electrokinetic chromatography with laser-induced fluorescence detection by Masatake Sano; Ikuko Nishino (80-83).
An assay for spermidine synthase (SPDS) activity in rat liver has been developed using micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection to enable the discovery of SPDS inhibitors. The assay was established by estimating the amount of spermidine (SPD) produced from the putrescine (PUT) present by SPDS. The SPD in an enzyme reaction mixture of homogenized rat liver could directly react with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) as a fluorescence derivatization reagent. The NBD derivatives of SPD and PUT could be separated and detected by MEKC-LIF detection within 15 min. The IC50 value measured for SPDS inhibitor, 4-methylcyclohexylamine, in rat liver by this assay was consistent with published data. Our SPDS assay using MEKC-LIF is simple and allows easy determination of SPDS activity in homogenized samples without troublesome procedures such as preparation of antibody or fluorescence-labeled substrate. The assay should be effective for discovering the SPDS inhibitors using biological samples.
Keywords: Polyamine; Spermidine synthase; Spermidine; Putrescine; Micellar electrokinetic chromatography; Laser-induced fluorescence detection; 7-Fluoro-4-nitrobenzo-2-oxa-1,3-diazone; Rat liver;
Quantitative determination of the anticancer agent tubeimoside I in rat plasma by liquid chromatography coupled with mass spectrometry by Ming-Jin Liang; Wei-Dong Zhang; Chuan Zhang; Run-Hui Liu; Yun-Heng Shen; Hui-Liang Li; Xiao-Lin Wang; Xiang-Wei Wang; Jian-Bao Zhu; Chun-Lin Chen (84-89).
Tubeimoside I is an important component isolated from Bolbostemma paniculatum. Tubeimoside I has been demonstrated to possess many pharmacological activities, including anti-inflammatory, antitumor, and antitumor-promoting effects. The purpose of the present study was to examine in vivo pharmacokinetics and bioavailability of tubeimoside I in rats by using a liquid chromatography coupled with mass spectrometry quantitative detection method (LC/MS). The plasma samples were deproteinated, evaporated and reconstituted in 100 μl methanol prior to analysis. The separation was performed by Waters Symmetry® C18 reversed-phase column (3.5 μm, 150 mm × 2.1 mm, Waters Inc., USA) and a SB-C18 guard column (5 μm, 20 mm × 4.0 mm). The mobile phase was a mixture of acetonitrile and water containing 5 μM NaAc (60:40, v/v). The method was validated within the concentration range 20–5000 ng/ml, and the calibration curves were linear with correlation coefficients >0.999. The lowest limit of quantitation (LLOQ) for tubeimoside I was 20 ng/ml in 0.1 ml rat plasma. The intra-assay accuracy and precision ranged from 92.4 to 104.9% and from 5.8 to 10.5%, respectively, while inter-assay accuracy and precision ranged from 94.2 to 95.0% and from 5.1 to 8.8%, respectively. The method was further applied to assess pharmacokinetics and oral bioavailability of tubeimoside I after intravenous and oral administration to rats. The oral bioavailability of tubeimoside I is only 0.23%, which indicates that tubeimoside I has poor absorption or undergoes acid-induced degradation. Practical utility of this new LC/MS method was confirmed in pilot pharmacokinetic studies in rats following both intravenous and oral administration.
Keywords: Tubeimoside I; Bioavailability; Pharmacokinetics; LC/MS;
Determination of cocaine and cocaethylene in plasma by solid-phase microextraction and gas chromatography–mass spectrometry by Iván Álvarez; Ana María Bermejo; María Jesús Tabernero; Purificación Fernández; Patricia López (90-94).
The present paper describes a method for the simultaneous determination of cocaine and cocaethylene in plasma. It was based in the extraction of the analytes by solid-phase microextraction (SPME), and gas chromatography–mass spectrometry (GC–MS) was used to identify and quantify the analytes in selected ion monitoring (SIM) mode. The method showed to be very simple, rapid and sensitive. The method was validated for the two compounds, including linearity (range 25–1000 ng/mL) and the main precision parameters. It was applied to ten plasma samples from cocaine and alcohol users, obtaining positive results in all cases.
Keywords: Cocaine; Cocaethylene; Solid-phase microextraction; Gas chromatography–mass spectrometry;
Analysis and comparison of active constituents in commercial standardized silymarin extracts by liquid chromatography–electrospray ionization mass spectrometry by James I. Lee; Mahesh Narayan; Jeffrey S. Barrett (95-103).
A sensitive method for the simultaneous quantitation of six active constituents in commercial silymarin standardized extracts was developed based on liquid chromatography (LC) in combination with mass spectrometry (MS). The six main active constituents, namely, silydianin, silychristin, diastereoisomers of silybin (silybin A and B), and diastereoisomers of isosilybin (isosilybin A and B) were completely separated and quantified by LC/MS. Silymarin obtained from Sigma–Aldrich Co. was evaluated and used as standard reference material for the six individual constituents in comparing the relative content of silymarin and the relative ratio of each constituent in commercial standardized silymarin extracts, respectively. Significant variation was found between different commercial silymarin sources. As a result, this method has proven useful in evaluating and quantifying the six active constituents in commercial milk thistle extracts. The calibration curves were over the range from 0.25 to 100 μg/mL for silychristin and silydianin, and from 0.10 to 100 μg/mL for silybin A, silybin B, isosilybin A and isosilybin B, respectively (r 2 ≥ 0.9958). For all six active constituents, the overall intra-day precision values, based on the relative standard deviation replicate for four QC levels, ranged from 1.18% to 12.4% and accuracy ranged from 89.4% to 112%. This methodology could easily be incorporated into standardized testing to assess content uniformity including lot-to-lot variation as part of routine process controls as well as a means to describe cross-product variation among the exiting marketed formulations.
Keywords: Silymarin; Milk thistle; Silybin; Isosilybin; LC/MS;
Sensitive quantification of diphemanil methyl sulphate in human plasma by liquid chromatography–tandem mass spectrometry by B. Do; S. Goulay-Dufaÿ; M.D. Le Hoang; N. Adoui; H. Graffard; F. Guyon; D. Pradeau (104-108).
A simple detection system with a high-performance liquid chromatography (HPLC) with positive ionisation-tandem mass spectrometry (ESI-MS/MS) for determining diphemanil methylsulphate (DMS) levels in human plasma using 4-diphemanylmethylene,1-methylpiperidine as an internal standard (I.S.), is proposed. The acquisition was performed with the multiple reactional monitoring (MRM) mode, by monitoring the transitions: m/z 278 > 262 for DMS and m/z 263 > 247 for the I.S. The method involved a simple single-step deproteinisation with acetonitrile. The analyte was chromatographed on a Zorbax® C18 reversed-phase chromatographic column by isocratic elution with 10−3 M ammonium acetate and 10−3 M hexafluorobutyric acid, adjusted to pH 7.0 with ammoniac/acetonitrile (40/60, v/v). The results were linear over the studied range (0.5–50.0 ng mL−1) and the total analysis time for each run was 10 min. The mean extraction apparent recoveries expressed at the 95% intervals of confidence were 94–104% for DMS and 92–106% for the I.S. The intra- and inter-assay precisions were 4.6–8.4% and 2.9–10.6%, respectively. The limit of quantification was 0.15 ng mL−1. The devised assay was successfully applied to the residual concentrations monitoring in infant.
Keywords: Diphemanil methylsulphate; HPLC; Tandem mass spectrometry; Quaternary ammonium; Plasma;
Determination of the dopamine agonist rotigotine in microdialysates from the rat brain by microbore column liquid chromatography with electrochemical detection by Jan Kehr; Xiao-Jing Hu; Takashi Yoshitake; Dieter Scheller (109-113).
Rotigotine, an investigational dopamine agonist formulated as a patch, is being studied in Parkinson's disease. A microdialysis technique, in combination with microbore column liquid chromatography and electrochemical detection, was developed to monitor rotigotine levels in the brain. Microdialysis probes were inserted into the striata of anesthetized rats, and samples were collected during perfusion with Ringer's solution. Rotigotine was separated using a C18 reversed-phase column. The mobile phase consisted of 50 mM Na2HPO4·2H2O, 2.5 mM sodium octyl sulfonate, and pH 4.5; 35% volume to volume acetonitrile. The flow rate was 30 μl/min, and the potential of the glassy carbon electrode was set to +850 mV. The method allowed monitoring of the time course of brain extracellular rotigotine levels with a detection limit of 1 nM following either intravenous (0.5 mg/kg) or subcutaneous (5.0 mg/kg) rotigotine injection.
Keywords: Rotigotine; Microdialysis; Electrochemical detection; Liquid chromatography;
Analysis of the sulphur mustard metabolites thiodiglycol and thiodiglycol sulphoxide in urine using isotope-dilution gas chromatography–ion trap tandem mass spectrometry by James Riches; Robert W. Read; Robin M. Black (114-120).
A sensitive method has been developed for the trace analysis of the sulphur mustard metabolite thiodiglycol (TDG) in urine, and its oxidation product thiodiglycol sulphoxide (TDGO) after reduction to thiodiglycol. Thiodiglycol was extracted from urine by solid phase extraction onto a polymeric cartridge and, after isolation, converted to its bis-heptafluorobutyryl derivative with heptafluorobutyryl imidazole. An ion trap mass spectrometer in selected reaction monitoring mode detected spiked concentrations down to 0.2 ng/ml with a signal to noise ratio > 3:1. Urine, from human volunteers with no known exposure to sulphur mustard, contained detectable but very low concentrations (<0.2 ng/ml) of thiodiglycol, consistent with previous observations using different methodologies. Combined concentrations of thiodiglycol and thiodiglycol sulphoxide were determined after reduction of the latter with titanium trichloride. In this case higher background levels (up to 3 ng/ml) were observed, consistent with the sulphoxide being the major excretion product of the two metabolites. The method was applied to urine samples, stored frozen for 13 years, from two casualties of accidental mustard poisoning. Levels of thiodiglycol were 1 and 3 ng/ml, which increased to 78 and 104 ng/ml after treatment of the urine with titanium trichloride.
Keywords: Sulphur mustard; Thiodiglycol; Thiodiglycol sulphoxide; Urine; Isotope-dilution; Gas chromatography; Ion trap tandem mass spectrometry;
High-sensitivity profiling of glycoproteins from human blood serum through multiple-lectin affinity chromatography and liquid chromatography/tandem mass spectrometry by Milan Madera; Yehia Mechref; Iveta Klouckova; Milos V. Novotny (121-137).
We report here the use of high-performance lectin affinity enrichment of glycoproteins at microscale levels using a series of silica-bound lectins. The potential of this approach is being demonstrated for the glycoprotein enrichment from microliter volumes of human blood serum. Individual injections of sample to the affinity microcolumns packed with four lectin materials with different glycan specificities (Con A, SNA-I, UEA-I, PHA-L), followed by off-line reversed-phase pre-fractionation and nano-LC/MS/MS, permitted identification of 108 proteins in the lectin-bound fractions spanning a concentration dynamic range of 7–10 orders of magnitude. In contrast, multi-lectin microcolumn affinity chromatography, an alternative enrichment approach allowed identification of only 67 proteins. An attractive feature of high-performance lectin affinity chromatography at microscale levels is the substantial reduction of sample losses that are commonly experienced with extensive sample preparation needed for larger sample volumes.
Keywords: Lectin; Multiple lectin; Affinity chromatography; Glycoproteins; Mass spectrometry; Human blood serum; High sensitivity;
Bioanalysis and pharmacokinetics of chitosan ester in rabbit serum by HPLC with postcolumn fluorescence derivatization by Yuhong Han; Zhihua Lv; Tingfu Jiang; Yuanhong Wang (138-142).
Interest in antiatherosclerotic activity of chitosan ester (PS916) with a new form of sulfate amino polysaccharide derived from marine chitin has necessitated the development of a sensitive and specific method to study its pharmacokinetics. A sensitive and reproducible high-performance liquid chromatography (HPLC) with postcolumn fluorescence derivatization method was developed and validated for the determination of PS916 in rabbit serum. Chromatography was carried out using a C8 reversed-phase column with an isocratic mobile phase consisting of methanol–water (20:80, v/v) at a flow rate of 0.2 ml/min. The derivatization procedure involved postcolumn reaction with guanidine hydrochloride in an alkaline medium at 110 °C. The fluorometric detector was operated at 250 nm (excitation) and 435 nm (emission). The assay was linear over the concentration range of 5–100 μg/ml. The lower limit of detection (LLOD) was found to be 1.0 μg/ml. The proposed method was successfully applied for a pharmacokinetic study of PS916 in rabbits.
Keywords: Polysaccharide; PS916; High-performance liquid chromatography; Fluorescence derivatization; Pharmacokinetic study;
A method for the identification of glycoproteins from human serum by a combination of lectin affinity chromatography along with anion exchange and Cu-IMAC selection of tryptic peptides by Ruiqing Qiu; Xiang Zhang; Fred E. Regnier (143-150).
This paper reports a method for identifying glycoproteins from human serum. Glycoproteins were selected with a concanavalin A (Con A) lectin column and then tryptically digested prior to sequential chromatographic selection of acidic and histidine containing peptides. Acidic peptides were selected with a strong anion exchange (SAX) column. Peptides captured by the SAX columns were then released and histidine-containing peptides in the mixture selected with a copper loaded immobilized metal affinity chromatography (Cu-IMAC) column. This serial chromatographic selection process reduced the complexity of proteolytic digests by more than an order of magnitude. Peptides selected by this serial process were then fractionated by reversed-phase chromatography (RPC) and identified by tandem mass spectrometry. The method was initially validated using human transferrin before application to human serum. The results show that all the peptides identified except one contained histidine and acidic amino acids.
Keywords: Glycoprotein; Lectin affinity chromatography; Ion exchange; Cu-IMAC;
Application of high performance anion exchange-pulsed amperometric detection to measure the activity of key sucrose metabolising enzymes in sugarcane by Peter L. Albertson; Christopher P.L. Grof (151-156).
A novel method using an HPAE-PAD system, which is routinely applied to detect carbohydrates at low levels (ng per sample injection), has been applied to the measurement of key sucrose metabolising enzyme activities in partially purified extracts of sugarcane tissues. Extraction and assay procedures tailored for the HPAE-PAD system enabled the accurate measurement of enzyme activities in more mature internodes than had previously been possible using enzyme coupled assay methodology. A major advantage of the HPAE-PAD method is the capability to monitor a broad range of sugars in each assay and provides an overarching perspective of the mix of competing enzymes that may be operating simultaneously in crude extracts. The technique has been successfully applied to measuring the activity of key sucrose metabolising enzymes in sugarcane stem tissue that is generally low in protein and high in endogenous sugars, primarily sucrose.
Keywords: HPAE-PAD; Enzyme activity; Invertase; Sucrose phosphate synthase;
Determination of plasma thiol bound to albumin using affinity chromatography and high-performance liquid chromatography with fluorescence detection: Ratio of cysteinyl albumin as a possible biomarker of oxidative stress by Yuki Ogasawara; Yoshio Mukai; Tadayasu Togawa; Toshihiro Suzuki; Shinzo Tanabe; Kazuyuki Ishii (157-163).
We examined the influence of oxidative stress on the relative amounts of various albumin-bound thiols in human plasma. To determine the ratio of thiols existing as mixed disulfides following oxidation, we developed a method combining fast purification of albumin using affinity columns and high-performance liquid chromatography (HPLC) with fluorescence detection for low molecular weight thiols which were labeled after reduction.When the effect of exposure of plasma to radical oxygen species on binding of thiols to albumin was determined by the present method, significant increases in the ratio of cysteine bound to albumin (Alb-Cys) to total cysteine were clearly demonstrated.
Keywords: Oxidative stress; Albumin mixed disulfide; Reactive oxygen species; Human plasma; Cysteinyl albumin; tert-Butyl hydroperoxide;
Method for the determination of blood methotrexate by high performance liquid chromatography with online post-column electrochemical oxidation and fluorescence detection by Hui Li; Wenhong Luo; Qingyu Zeng; Zhexuan Lin; Hongjun Luo; Yuan Zhang (164-168).
Methotrexate (MTX) has been widely used at low dose for the treatment of different diseases including rheumatoid arthritis. MTX might be present in plasma in free form, and in blood cells in methotrexate polyglutamate (MTXPG). A rapid and sensitive HPLC method was developed for the determination of plasma MTX level, whole-blood MTX level, and whole-blood total MTX (MTX + MTXPG) level. To determine plasma MTX level or whole-blood MTX level, a 0.2-ml aliquot of plasma or whole blood (after a freeze–thaw cycle to break blood cells) was well mixed with 0.8 ml methanol and centrifuged. To determine whole-blood total MTX level, a 0.1-ml aliquot of whole blood (after a freeze–thaw cycle) was mixed with 80 μl ascorbic acid (114 mM) and incubated at 37 °C for 2 h to enzymatically convert the MTXPG to MTX. Then 20 μl NaOH solution (0.5 M) and 0.8 ml methanol were added and mixed well. After centrifugation, a 0.5-ml aliquot of the supernatant was evaporated to dryness and re-dissolved in 0.2 ml hydrochloric acid (10 mM). Methylene chloride (0.2 ml) was added and mixed well. After centrifugation, the top aqueous layer was injected to HPLC for analysis. After the MTX was eluted from the HPLC column, it was electrochemically oxidized and detected by a fluorescence detector. Recoveries of spiked MTX at ppb (ng/ml) level were between 87.9 and 118% with within-day relative standard deviation less than 5.2% and day-to-day relative standard deviation less than 9.8%. The limit of detection (LOD) and limit of quantitation (LOQ) of the described method were 1.2 and 2.6 ng/ml, respectively.
Keywords: Methotrexate; HPLC; Online electrochemical oxidation;
Determination of metolazone in human blood by liquid chromatography with electrospray ionization tandem mass spectrometry by Guangli Wei; Shuhua Xiao; Changxiao Liu (169-173).
A rapid, sensitive and accurate liquid chromatographic–tandem mass spectrometric method is described for the determination of metolazone in human blood. Metolazone was extracted from blood using ethyl acetate and separated on a C18 column interfaced with a triple quadrupole tandem mass spectrometer. The mobile phase consisting of a mixture of acetonitrile, 10 mmol/l ammonium acetate and formic acid (60:40:0.1, v/v/v) was delivered at a flow rate of 0.5 ml/min. Electrospray ionization (ESI) source was operated in positive ion mode. Selected reaction monitoring (SRM) mode using the transitions of m/z 366 → m/z 259 and m/z 321 → m/z 275 were used to quantify metolazone and the lorazepam (internal standard), respectively. The linearity was obtained over the concentration range of 0.5–500 ng/ml for metolazone and the lower limit of quantitation (LLOQ) was 0.5 ng/ml. For each level of QC samples, inter- and intra-run precision was less than 8.07 and 3.56% (relative standard deviation (RSD)), respectively, and the bias was within ±4.0%. This method was successfully applied to the pharmacokinetic study of metolazone formulation after oral administration to humans.
Characterization of enantioselective binding of racemic natural tetrahydropalmatine to DNA by chromatographic methods by Xingye Su; Feng Qin; Liang Kong; Junjie Ou; Chuanhui Xie; Hanfa Zou (174-179).
A racemate from natural product, tetrahydropalmatine (THP), was characterized on its enantioselective binding to DNA by the chromatographic methods including microdialysis/HPLC, centrifugal ultrifiltration/HPLC and immobilized DNA affinity chromatography. It was found that its (+)-enantiomer was preferential to binding on B-form duplex DNA including calf thymus DNA, AT and GC sequence oligo DNA, as well as triplex oligo DNA. The binding constants of the THP enantiomers to ct-DNA were determined with the methods of microdialysis/HPLC and frontal affinity chromatography. In addition, the DNA structural preference of either enantiomer was evaluated with the chromatographic methods.
Keywords: Tetrahydropalmatine; Enantioselectivity; DNA binding; Natural products; Chromatographic methods;
Simultaneous determination of methamphetamine and its metabolite, amphetamine, in urine using a high performance liquid chromatography column-switching method by Mitsuru Kumihashi; Kiyoshi Ameno; Takayuki Shibayama; Keisuke Suga; Hiroshi Miyauchi; Mostofa Jamal; Weihuan Wang; Ikuo Uekita; Iwao Ijiri (180-183).
We describe here a simple, precise, and highly sensitive method for the simultaneous determination of methamphetamine (MA) and amphetamine (AM) in urine using a high performance liquid chromatography (HPLC) column-switching method. A PK-2A (Shodex) column was used for extraction and deproteinization, and a CAPCELL PAK SCX semi-micro, polymer-coated cation-exchange column was employed for separation. The urine sample was mixed with an equal volume of borate buffer (0.1 M, pH 9.4), and then 100 μl of the mixture was injected into the HPLC column. The column was switched for 6 min, and then 10 min later detection was performed at 210 nm. Recovery yields of the MA and AM spiked in the urine were 93.0–100.4% with a coefficient of variation of less than 1%. The calibration curves of MA and AM were in the range of 0.1–10 μg/ml with good linearity (r 2 = 0.999), with the limit of qualification being 0.005 μg/ml. This method of using HPLC with column-switching can be used for both qualification and quantification of MA and its metabolite, AM, in urine, especially in forensic cases.
Keywords: HPLC; Column-switching; Methamphetamine; Amphetamine; Urine;